Six months have passed since the victorious Battle of Britannia and the reclamation of Gallia from Neuroi invaders. Yoshika Miyafuji, member of the famed 501st Joint Fighter Wing, has come back home to Fuso and graduated from middle school. However, her fight is far from over. She receives a letter supposedly sent by her long-deceased father, containing blueprints of a state-of-the-art Striker Unit he had been working on before his death. The Unit, designed specifically for Yoshika, might be capable of harnessing her extraordinary magical powers. Meanwhile, a new threat in Europe is rising. A Neuroi nest of an unprecedented size and might has appeared over Venezia, wiping out local Witch forces and instantly swallowing the northern part of the country. To make matters worse, a newly spotted humanoid type of Neuroi is attempting to come into contact with humans. Yoshika, incapable of abandoning her friends on the front lines, must once again venture to the war-torn continent.

After fending off the threat of a Neuroi invasion of Romagna and destroying the enemy's nest over Venezia, Yoshika Miyafuji goes back to her home town in the Empire of Fusou. Despite the loss of her magical and healing abilities, the former officer of the 501st Joint Fighter Wing wants to continue studying medicine. This is in order to help those in need, both civilians and those on the front lines alike. She receives an invitation from a prestigious school in Europe and decides to accept the offer, embarking on a journey back to the war-torn continent. However, a new danger arises, as more and more Neuroi manage to break through human defense lines, appearing unnoticed in supposedly safe areas and disturbing the balance of power in Europe. Yoshika's sense of duty collides with her inability to pilot a Striker Unit, but with her friends in peril, it seems that there is only one thing she can do.

Preparations for a new offensive against the Neuroi—a mysterious race of alien invaders—are well underway. The objective is securing Berlin, the capital city of the Empire of Karlsland, which is necessary for wiping out the Neuroi threat from Europe. However, as the enemy is capable of adapting to the battlefield on a daily basis, the allied forces and the current state of Striker technology might not be enough to achieve a victory. Meanwhile, Yoshika Miyafuji, a Witch from Fuso, continues her medical studies in Lausanne. Having recovered from a recent incident that deprived her of magical power, she is eager to assist in the war effort. The call to arms soon arrives and the scattered witches of the 501st Joint Fighter Wing must be gathered once again for a final push against the enemy.

In the year 1939, humanity was on the verge of destruction. Equipped with weapons of devastating power, a mysterious alien race, known as the Neuroi, began an invasion of the Earth, ravaging the planet, obliterating countries, and vanquishing human armies. To counter this seemingly unstoppable force, the world's powers united. A new device was produced, called the Striker Unit, capable of facing Neuroi technology. Girls equipped with the Striker Unit became known as the Witches, forming humanity's last line of defense. They began their fight against the Neuroi, utilizing their now-enhanced magical abilities, flaunting destructive weapons no human could manage. Fast forward to 1944—Yoshika Miyafuji is a young teenage girl who eventually decides to join the 501st Joint Fighter Wing, known as the Strike Witches. With no formal training however, can Miyafuji learn to cooperate with her fellow Strike Witches? And if so, can she save humanity from the Neuroi onslaught?