In 1939, after the world has been devastated by an invasion of the "Neuroi"—alien war machines of unknown origin—humanity continues to struggle in an uneven fight. With no conventional arms able to pierce through the aliens' hardy armor, resistance forces spare no expense in their search for a way to level the playing field.

Eventually, the so-called "Striker Unit" is designed—a state-of-the-art device utilizing the innate magical powers of young girls and turning them into deadly weapons. The women who are now gathering from all around the world to fight for the survival of the human race are known as "Witches," with the 501st Joint Fighter Wing—the "Strike Witches"—standing at the forefront of the deadly conflict.

Six months after the battle in Venezia, Minna, Gertrud, and Erica are stationed at Saint-Trond Base in Belgica, joined by Karlsland witch Heidemarie. Ursula, Erica's twin, delivers a 50mm cannon and Jet Striker Unit, with Gertrud chosen to test them—despite Erica's concerns from a previous mishap. Erica later tests a Twin Striker with Ursula, but her impatience causes failure, leading to harsh words that dishearten Ursula. When Minna and Gertrud are ambushed by splitting Neuroi, Gertrud tries using the 50mm cannon but is overwhelmed. Hearing Minna in danger, Erica takes the Jet Striker and cannon herself, struggling to control them until Ursula arrives to help. Together, the sisters save Minna and defeat the Neuroi.

Learning that island of Delos has been taken over by Neuroi, Charlotte E. Yeager and Francesca Lucchini are teamed up with Hanna-Justina Marseille and her partner Raisa Pöttgen to draw out the Neuroi hiding within it. Francesca, who has strong family ties with Delos, objects to their superior's plan to destroy the island, so they work out a new plan to have Hanna and Raisa draw out the Neuroi while Charlotte and Francesca drop bombs on it. When this plan ends up failing, Francesca tries to attack on but is inevitably forced to retreat. The next day, before the bombardment on the island is due to take place, Charlotte, unwilling to let Francesca down, decides to make use of an abandoned ship's winch to pull the Neuroi out of its hiding place, allowing it to be destroyed without any damage to the island. Afterwards, Francesca takes Charlotte and the others on a tour of Delos and its artifacts.