The year is 1939 - it was then that the Neuroi appeared. Nobody knows where they came from or what their ultimate agenda is, but the fact remains that their attacks drove people out of their towns and cities. In order to take arms against them, humanity develops a new anti-Neuroi weapon called the "Striker Unit." Using the power of magic to fight against the monsters, this new device enhances and amplifies the power of female magic-wielders. To use this device, young witches from all over the world have been brought together to form an elite task force unit called the 501st Joint Fighter Wing, commonly known as the "Strike Witches."

After discovering that the Neuroi are capable of communication with humans and making peace with the nest above Gallia, the Witches squads are on a mission to reestablish communication, when a sudden attack causes the Neuroi to vanish. The Witches discover a new and massive nest that just appeared, covering almost all Europe. Ruthlessly and without mercy it annihilates allied forces along with Witches. Word of the attack reaches Fuso and a support battalion is deployed to rescue the survivors. Among the rescue team is the former Striker captain Mio Sakamoto and the now civilian Yoshika Miyafuji, who want to save their dear friends.

After disbanding, the girls of the 501st Joint Fighter Wing return to face the threat of the Neuroi again, now joined by a new member, Shizuka Hattori.