The story starts off in a New York setting sometime after the first movie, presumably between 1886 and 1889,
and has Fievel recalling a strange dream in which he and his family went west (a way of dismissing, or forshadowing the second American Tail movie, which underperformed at the box office).
Fievel and Tony discover that an ancient treasure lies underneath Manhattan when snooping around an abandoned subway (the Beach Pneumatic Transit system) and stumbling upon the remains of a dead mouse clutching a treasure map, deciding they must find it with the help of an archaeologist Tony knows: Dr. Dithering, along with fighting five villains as well.
The movie focuses on the relationship between the over-exploited workers of a sweatshop (in this case, a cheese production line) and the factory’s rich owners: Mr. Grasping (Ron Perlman), Toplofty (Tony Jay) and O’Bloat (Richard Karron). It also focuses on the plight of the Native Americans in the United States.
The treasure under Manhattan turns out to be a group of Lenape mice living a long distance beneath the surface (far below the sewers, riding in an underground pressurized train) that decided to hide when they saw how the first Europeans only brought war and disease with them and didn’t want to wait for the European mice to do the same to them.
An emotional scene ensues when Fievel must struggle with how cruel his own people the Europeans were (and still are at the time the film takes place) to the natives of America.
The sachem decides to send his daughter, Cholena, to the surface to see if they have “changed their ways.” Upon their return, Scuttlebutt (one of the members of the expedition to find the treasure) reports to the villains unbeknownst to the rest of the members of the expedition, who then decide to use this to their advantage.
The mouse NYPD Chief, McBrusque (Sherman Howard) and McBrusque’s henchman/Dr. Dithering’s associate, Scuttlebutt (John Kassir), who also report to the villains, engage in a bout of police brutality, burning down “every mouse house and rat hole” until they find her.
After the angry mouse mob try to capture Cholena and anyone else involved with her, Fievel and his friends decide to take Cholena back underground, but the police find out and go after them. Meanwhile, everyone finds out about Dr. Dithering’s friendship with the Indian and take him to the butcher shop for his punishment.
Papa tells everyone about how madness like this is why they all left for America and should work together to become friends with those different from them as the fellow Americans they are. Upon returning Cholena to her home and telling the chief what is happening, he gives them a bomb to collapse the tunnel connecting the Native Americans to the outside world. This floods it, together with the evil McBrusque and Scuttlebutt as they fall into a pit.
The movie ends with Fievel’s papa forming a worker’s union and the villains agreeing amongst themselves to negotiate “with that riff-raff” because otherwise, they’ll go on strike and make them go bankrupt while Tiger the cat, who is now the new police chief, watches them.
The last scene is Fievel seeing, through a foldable telescope, Cholena and her father disappearing into a hidden door at the foot of a statue, which pleases Fievel.