A widowed field mouse named Mrs. Brisby lives with her family within a cinder block in a field belonging to the Fitzgibbon family. and is preparing to move before the plowing season begins. Unfortunately, her youngest son, Timothy, has fallen ill. She visits Mr. Ages, another mouse and friend of her late husband Jonathan, who diagnosis Timothy with pneumonia and gives her medicine for him. On her way home, she meets a crow named Jeremy tangled up in string that he was preparing for a nest in hopes of attracting a mate. While freeing him, the pair are ambushed by the farmer's pet cat Dragon and barely escape. Mrs. Brisby briefly loses the medicine, which Jeremy retrieves and returns to her. She returns home just as her nosy neighbor, Auntie Shrew, is leaving following an argument with her son, Martin. Auntie Shrew had come by to tell her of Moving Day and to prepare moving her family. After reprimanding her children, particularly Martin, for their behavior, she gives Timothy his medicine.
The next day, Mrs. Brisby comes across Auntie Shrew, who tries persuading her to get out quickly just as Mr. Fitzgibbon starts his tractor up, causing the Shrew to run off and begin warning the other animals to run away. Determined to save Timothy, who is resigned to being in bed and sleeping at the moment (Mr. Ages warned not to bring him outside for three weeks, lest he catch his death of cold), Mrs. Brisby leaves her children with the Shrew and attempts to stop the tractor. The Shrew follows in order to aid her, and the plan proves successful. Both flee and the Shrew suggest going to see the Great Owl on what to do. Mrs. Brisby is reluctant to do so, considering owls eat mice, but goes to his hollow anyway via riding on Jeremy and enters his hollow. The Great Owl only tells her to move her house; upon learning that she is Jonathan Brisby's widow, however, he tells her to visit the rats living in the rose bush next to the farm house and ask for their leader, Nicodemus, and to move her house to the protected side of the stone. He leaves afterwards.
Taking the Owl's advice, Mrs. Brisby goes to the rose bush and assigns Jeremy with protecting her children. Upon entering, she is chased off by a rat named Brutus, who guards the inside. She meets Mr. Ages, now with a broken leg, and tells her of her meeting with the Great Owl. Mr. Ages agrees to take her to Nicomedus. Along the way, they meet Justin, the captain of the guard. They both enter a council meeting, where Jenner, a fellow member of the rats, is opposing Nicodemus' position as leader. Mr. Ages and Jonathan introduce Mrs. Brisby to the rats and that the Great Owl told her to seek their help in moving her house. Jenner openly claims to agree to it, though secretly planning to use it for his own needs.
Mrs. Brisby then meets with Nicodemus, who reveals that Jonathan was a life-time friend to the rats. After making her read from a book documenting Jonathan, which reveals his death following an incident with Dragon, Nicodemus explains that the rats once lived in the streets but were eventually captured and taken to a place called NIMH, where they were tested on and injected with serum; the result increased their intelligence, giving them the ability to read and unlock cages and doors. All of the rats escaped, along with mice and climbed out through a shaft, and all but two of the mice, Jonathan and Mr. Ages, were blown away. Jonathan was responsible for helping them escape. Having finished the story, Nicodemus gives Mrs. Brisby an amulet which Jonathan had made for her. The stone in it glows red when its wearer shows true courage. The pair then depart to move Mrs. Brisby's house. Meanwhile, out of sight, Jenner reveals to his partner Sullivan that he intends to make the house fall during the move, hoping that it will kill Nicodemus and leave them to stay in the rose bush permanently.
Nicodemus and Justin reveal to Mrs. Brisby that they intend to leave the rose bush and move to a place known as Thorn Valley, where they can live freely without having to steal electricity from the farm house, which is how they managed to thrive for so long. To make the moving of her house safer, though, they must first drug Dragon's food and put the cat to sleep (Jonathan had been killed in a previous attempt, and Mr. Ages had broken his leg in another). Mrs. Brisby volunteers to drug the cat and briefly reunites with Jeremy, who is on the run from Auntie Shrew after she tied him up, and tells him to bring back his collection of string in hopes of using it to help move her house.
She enters the farm house through a hole in the floor and successfully drugs Dragon's food before he is let inside, but is unfortunately caught by the family son, Billy, who plans to keep her as a pet. Justin is forced to leave and help with the move, promising to come back for her later. Later, Mrs. Brisby overhears Mr. Fitzgibbon contacting NIMH and permitting them to bulldoze the rose bush and exterminate the rats, who are just beginning to move the house. Unfortunately, the children and Auntie Shrew are still inside, the latter accidentally getting knocked out with a hit to the head. Taking advantage of the situation, Jenner puts his plan into action and begins cutting the ropes holding the house up. The ploy works, and Nicodemus is crushed and killed in the collapse, which leaves the house trapped in the mud. Mrs. Brisby finally manages to escape and reunites with them, and Mr. Ages informs her of Nicodemus' death. She warns them of NIMH arriving in the morning and that they must leave immediately. Angered at the possibility of the rats siding with her, Jenner calls her a hysterical liar and attacks her. Sullivan alerts Justin of the situation, and the latter comes to Mrs. Brisby's aid as Jenner attempts to take the amulet off her neck. Jenner mortally wounds Sullivan and clashes with Justin in a sword duel, in which he admits to killing Nicodemus, which ends with Justin stabbing him in the torso. While Justin is distracted and convincing the rats to leave for Thorn Valley, Jenner makes one final attempt to kill him, but Sullivan kills him by throwing a knife into his back, saving Justin's life before succumbing to his own wounds.
Meanwhile, Mrs. Brisby's house begins sinking into the mud. The rats all attempt to save it whilst the children inside try to stay above the mud seeping inside. As the house completely sinks under, Mrs. Brisby's courage and determination to save her family powers the amulet, giving her the power to rise the house out of the ground and move it safely. She then passes out from the weight of the power.
Some time later, the family has completely settled down and Timothy is beginning to recover. Jeremy, who has passed by with the string he was asked to get, begins feeling useless now that the house is already moved; he depression turns to joy, however, upon meeting a female crow who is as clumsy as he is. He then asks Mrs. Brisby for the amulet (he was hypnotized by it in their previous encounter) in hopes of impressing "Ms. Right" as he calls her, but Mrs. Brisby tells him that she has already given the amulet to Justin, the new leader of the rats who have since departed for Thorn Valley, and to just be himself in order to impress her, which he agrees to. The Brisby family prepare for another visit from Auntie Shrew as they begin hoping of visiting Thorn Valley, and Jeremy and his newfound mate hold opposite sides of a piece of string and fly off together into the sunset.
- Elizabeth Hartman as Mrs. Brisby
- Derek Jacobi as Nicodemus
- Hermione Baddeley as Auntie Shrew
- John Carradine as The Great Owl
- Dom DeLuise as Jeremy the Crow
- Arthur Malet as Mr. Ages
- Peter Strauss as Justin
- Paul Shenar as Jenner
- Aldo Ray as Sullivan
- Shannen Doherty as Teresa Brisby
- Wil Wheaton as Martin Brisby
- Ian Fried as Timothy "Timmy" Brisby
- Jodi Hicks as Cynthia Brisby