Six months after the battle over Brittania, the 504th Joint Fighter Wing of Strike Witches, while on a mission to make contact with the Neuroi, encounter an even greater Neuroi nest hovering over Venesia which wipes out the previous Neuroi and attacks relentlessly. Meanwhile, Yoshika, who had just graduated from middle school, receives a letter from Amaki Suwa, containing blueprints supposedly sent by her father. When Yoshika goes to deliver these to Mio, she gets word that the stronger Neuroi had overwhelmed the 504th Joint Fighter Wing and that Lynette and the others are in danger. Yoshika is told by Mio to go home, but when she notices Mio heading into battle again, Yoshika takes her old Striker Unit and follows after her. Admiring her spirit, Mio allows Yoshika to accompany her. However, a week into their flight, their plane comes into contact with a Neuroi ship.

As the Neuroi continues to attack the plane, a fleet of battleships provide support, but are easily defeated by the Neuroi. With Mio still unable to use her shield, Yoshika flies into battle to protect the ships with her enhanced magic. Mio then uses her enhanced katana to slice through the Neuroi's beam and destroy it. However, it soon reforms itself, its core continuously shifting places to avoid being destroyed. Yoshika has a tough time facing it until Shirley, Lucchini, Lynette and all the other Witches arrive to assist, giving Mio the opportunity to destroy it with her katana. Setting up base in Romagna, Minna officially reforms the 501st Joint Fighter Wing.

Mio meets up with Junko Takei of the 504th Joint Fighter Wing to learn about the new breed of Neuroi. Having gotten out of shape following inactivity during the past six months, Yoshika, Lynne and Perrine are sent to an old lady named Anna Ferrara, who teaches them the basics, namely how to ride old fashioned brooms. They struggle at first, but they soon start to get the hang of it. During their training, a Neuroi ship threatens to come into contact with Anna's home, but the girls team up to defeat it.

The Witches obtain a prototype Jet Striker model from Karlsland. After an argument between Shirley and Barkhorn, Barkhorn gets to use it, as Lucchini convinces Shirley not to use it after trying it herself. Barkhorn relishes in the Jet Striker's power and outperforms Shirley in a series of performance tests, but it ends up draining too much of her magic, causing her to faint midflight. Minna prohibits anyone from using the Striker as a result, and confines Barkhorn to her quarters. She breaks the ban anyway when a fast Neuroi appears. Although she defeats the Neuroi, Barkhorn faints as, this time, the Jet Striker goes out of control, forcing Shirley to push her Striker Unit to its limits to save her, destroying the prototype in the process. Later, Erica's twin sister, Ursula, pays them a visit, with a shipment of potatoes as a token of apology for the flawed prototype.

While Yoshika, Shirley and Lucchini are in Romagna's capital, Roma, buying supplies and gifts, Lucchini spots a girl named Maria being accosted by some suited men and rescues her. The two quickly become friends and spend the day exploring the city, although Lucchini ends up spending all the food money Shirley gave her. When a Neuroi ship appears, Lucchini rejoins Yoshika and Shirley to defeat it. Afterward, Lucchini treats Maria to a bird's eye view of Roma, telling her the importance of protecting it. After they return home, it is revealed that Maria is actually the Duchess of the Principality of Romagna, who gives her thanks to Lucchini during her inauguration by airdropping food supplies to the Witches.

The Witches discover a huge Neuroi spire whose core is in a location far beyond the altitude that their Striker Units can reach. Formulating a plan using rocket boosters in stages to reach the required altitude, Sanya is chosen to attack the core. Having never needed to use a shield in combat, Eila becomes furious when Yoshika is chosen to protect Sanya instead of her. Eila tries to get Perrine to train her to use shields, but to no avail. Sanya becomes annoyed with Eila's willingness to give up and gets into a fight with her. The next day, as the Witches launch Sanya and Yoshika to the stratosphere, Eila apologizes and expresses her desire to protect Sanya. Seeing this, Yoshika decides to switch places with her. Eila finally uses her shield to protect Sanya as she destroys the core.

After enjoying the newly-installed bath, a strange bug that Lucchini found earlier in the morning is embarrassing the Witches by literally getting into their pants, as well as causing electrical outages around the base. They soon realize that the bug is the core of a Neuroi ship that is sapping electricity from the base, disabling the alarm system, as it feeds a larger unit lurking near the base. After the Witches ensue a lengthy chase for the bug around the base, it is Minna who inadvertently destroys the Neuroi bug when it crawled inside her pants, destroying the larger unit as well. It is technically Minna's 200th Neuroi kill, and she was awarded a medal by the Luftwaffe for the milestone. (from wikipedia)

Mio trains to obtain the true Reppuzan, and refuses to teach it to Yoshika despite her requests. Later, during a practice session with Perrine, Yoshika starts to have some trouble flying. Mysteriously, though, Yoshika is given a clean bill of health, and her Striker Unit is in perfect running condition. The next day, Yoshika and Lynnette are sent to the battleship Yamato to heal crew members injured in a lab explosion. When the Yamato and its battle group is attacked by a blimp-shaped Neuroi ship, Yoshika is unable to fly, leaving Lynette alone to defend the battle fleet while they retreat. As Yoshika laments her inability to help, she hears her father's voice and is presented with a new Striker Unit, which was developed based on his research findings. Now able to use her powers to the maximum with a Striker Unit built just for her, Yoshika manages to save Lynette just in time and defeat the Neuroi single-handedly.

While visiting Gallia, Perrine learns that children can't get to school because the bridge is broken. This preoccupies her, even thinking of selling the last family heirloom to fund the bridge's repairs. On a training trip at the beach, Lucchini finds a treasure chest, which Perrine salvages in the hope of finding treasure. After going through an endless number of chests, they find a puzzle chest containing a map, which Perrine, Yoshika, Lynnette and Lucchini follow to an underwater cave filled with booby traps. Meanwhile, while looking for them, Mio and the others encounter a trap which causes Mio to become drunk, kissing Minna and running off. Perrine's group comes across a room with a statue which Perrine defeats when it attacks them. They eventually find the treasure, which is nothing more than a herb and spice garden. However, a slightly hungover Mio reassures Perrine that it's the feelings that people truly treasure. After returning to base, Perrine receives a letter from Gallia informing her that the children worked together to build a new bridge.

Mio progresses in perfecting the true Reppuzan, but it drains much power from her. After Minna proposes an operation to attack a Neuroi base, the top brass send in Captain Hanna Marseille of the 31st Karlsland Luftwaffe Storm Witches to join the mission. Since Barkhorn and Hanna don't get along, Erica volunteers to accompany her on the mission. After fooling around in a training session, Hanna and Erica are ordered to stay in a room together to learn to cooperate. Hanna is obsessed with winning, and has always wanted to settle things with Erica, whom she views as her equal, but as of late she is annoyed with Erica's lack of competitive spirit. But when Hanna badmouths Barkhorn, who wanted an autograph for her sister, Erica agrees to compete with her during the mission. When they reached a draw after destroying the Neuroi, they have a tiebreaker dogfight, also ending in a draw. Before she leaves, Hanna decides to give the autograph.

Mio still experiences power drains from her use of the true Reppuzan. Later, she scolds Yoshika for touching her sword--which reacted to her powers and nearly caused her to faint. Later, Mio and Minna receive orders from their superiors for a final attack on the Neuroi nest. They plan to defeat it by powering the Yamato with Neuroi technology. The mission's failure would mean the surrender of Romagna and the disbanding of the 501st Strike Witches. Mio becomes determined to complete the True Reppuzan, but Minna, aware that her sword has drained nearly all of her magical power, stops her--which Yoshika overhears. The next day, they begin the charge and Mio's Reppuzan fails to work, much to her despair. Meanwhile, the Yamato activates its Neuroi Dynamo, allowing it to fly and repair itself. But when the Neuroified Yamato hits the hive bow first, its Neuroi Dynamo stopped working, paralyzing its firing controls. With the mission deemed a failure, Mio, never giving up, charges towards the Yamato to attempt to restart the Dynamo with her remaining magic power.

Despite Minna's objection, Mio heads inside the Yamato and gives the Neuroi Dynamo her remaining magic power which kick-starts the engine. Mio is then able to fire the main cannon, destroying the Neuroi nest. It is revealed however that the dome was mearly a shield concealing the hive's massive core, which Mio becomes absorbed into. Not giving up, Yoshika, with backup from the other Witches, retrieves the Reppumaru and is able to perform the True Reppuzan which only Fuso Witches are capable of performing. This destroys the giant Neuroi core at the cost of Yoshika's remaining magical power, freeing Mio in the process. With Venezia finally free from the Neuroi, the 501st Strike Witches are once again formally disbanded; and Yoshika begins her medical career. The final shot depicts Yoshika's special Striker Unit having washed up on a beach alongside the Reppumaru, the shadow of an unknown individual then appears to find them, setting the scene for the film.