Mrs. Brisby, a timid widowed field mouse, lives in a cinder block with her children in a field on the Fitzgibbons' farm. She prepares to move her family out of the field as plowing time approaches, but her son Timothy has fallen ill. She visits Mr. Ages, another mouse and friend of her late husband, Jonathan, who diagnoses Timothy with pneumonia and provides her with medicine. Mr. Ages warns her that Timothy must stay inside for at least three weeks or he will die. On her way home, she encounters Jeremy, a clumsy but friendly crow. They both narrowly escape from the Fitzgibbons' cat, Dragon.
The film begins with a prophecy from the first film, telling how one of Jonathan and Mrs. Brisby's sons would save Thorn Valley from "the secret of NIMH". Timothy is chosen to go, but his older brother Martin believes he should have been the one chosen. Martin decides to prove his quality, and goes off to find his own adventure.