The film, although a sequel, was created without Don Bluth's support or input. The film is unrelated to Racso and the Rats of NIMH, the sequel to the book on which the original film was based.
The film begins with a prophecy, telling how one of Jonathan's 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. One day, years later, Timmy comes across a very beautiful female field mouse named Jenny McBride, whose parents were two of the lost six mice who tried to escape NIMH but were thought to have died. The Thorn Valley council decides it would be too dangerous to save the nine mice, so Jenny and Timmy go alone. They take a hot air balloon, but it is attacked by a  and crashes in the forest. Seeking help, they visit the Great Owl. They learn that a caterpillar named Cecil has teamed up with Jeremy, the crow from the first film, to trick the forest animals into paying them money. Jeremy and Cecil take the two mice to NIMH where they find two of the rats from Thorn Valley, Justin and Brutus. They also learn that Dr. Joseph Valentine, the head scientist, has made Martin insane. After being made insane, Martin took over the lab and brainwashed Dr. Valentine with a device similar to the one Valentine used on him and made him think he was a dog. Martin plans to use an army of lab rats riding a flock of ravens to take over Thorn Valley. He has even turned two cats crazy, which catch the others.
He asks Timmy to join him, but he refuses and is locked in a cage as Martin drags Jenny away to make her his queen. Timmy escapes with the help of Cecil and they go to free Jenny. Together, they knock out Martin and Timmy tricks Martin's army of ravens and rats to fly in the wrong direction. Timmy and Jenny then leave to find the others, only to find that NIMH is on fire. The survivors (Timmy, Jenny, and her parents) flee. Timmy goes back to save Martin. Before he leaves, Jenny tells him she loves him and he tells her the same. After being attacked by the crazy cats once again, Timmy sends them down an elevator shaft and finally finds Martin. Then the lab is destroyed by flames.
Timmy and Martin are able to escape through the lab's skylight, with some help from Jeremy. Jeremy takes the survivors to safety. The mice return to Thorn Valley, Martin returns to normal and Timmy is now a hero. How Martin turned normal is left out of the film, but the device that turned Martin crazy could have been temporary and since Martin was shown doing it on himself several times this could be the reason. A likeness of Timmy is added to the statue of Jonathan as the people of Thorn Valley cheer. Then the film ends
- Ralph Macchio - 17 year old Timmy Brisby
- Dom DeLuise - Jeremy
- William H. Macey - Justin
- Arthur Malet - Mr. Ages
- Hynden Walch - Jenny McBride
- Andrea Martin - Muriel
- Harvey Korman - Floyd
- Meshach Taylor - Cecil
- Phillip Glasser - Redeemed 19 year old Martin Brisby
- Eric Idle - Evil 19 year old Martin Brisby
- Phillip Van Dyke - Young Martin Brisby
- Andrew Ducote - 10 year old Timmy Brisby
- Alexander Strange - 13 year old Timmy Brisby
- Debi Mae West - Mrs. Brisby
- Doris Roberts - Auntie Shrew
- Steve Mackall - Dr. Joseph Valentine
- Darlene Carr - Helen
- Jamie Cornin - Teresa Brisby
- Whitney Claire Kaufman - Cynthia Brisby
- Jameson Parker - Troy
- Kevin Michael Richardson - Brutus
- Jim Cummings - Guard #1
- Frank Welker - Killer the Bulldog, Snake and Hawk
- Besides respecting the history of NIMH, the ending is hated by the majority of the audience. The changing of fan favorite Mrs. Brisby to Timmy makes this sequel one of the most unpopular Don Bluth films, thus making Timmy one of the most unpopular Don Bluth characters, making it one of the few Don Bluth sequels to be explicitly dismissed from canon.