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In an abandoned river boat in Devil's Bayou, a young orphan named Penny drops a message in a bottle containing a plea for help into the river. The bottle washes up in New York City, where it is recovered by the Rescue Aid Society, an international mouse organization inside the United Nations. The Hungarian representative, Miss Bianca, volunteers to accept the case and along with Hubie, Marina, Rocko, Splatter, Petra, Beanie, Timmy, Pikatwo, Bonkers, Fawn and Buck, they choose Bernard, a stammering janitor, as a co-agent. The team visit Morningside Orphanage, where Penny lived, and meet an old cat named Rufus. He tells them about a woman named Madame Medusa who once tried to lure Penny into her car and may have succeeded in abducting Penny this time.

On the way to Medusa's pawn shop, the team are stopped abruptly by an unexpected visitor, Stanely the Troll. He explains that he had heard of their mission and has agreed to help them.

The team travel to Medusa's pawn shop, where they discover that she and her partner, Mr. Snoops, are on a quest to find the world's largest diamond, the Devil's Eye. They also discover that Medusa and Mr. Snoops are at the Devil's Bayou with Penny, whom they have indeed kidnapped, and whom they guard with two trained alligators called Brutus and Nero. With the help of an albatross named Orville, and a dragonfly named Evinrude, the mice and our heroes follow Medusa to the bayou. There, they learn that Penny was captured to enter a hole that leads down into the pirates' cave where the Devil's Eye is located.

Bernard, Miss Bianca, Hubie and his friends find Penny and devise a plan of escape and send Evinrude to alert the local animals, who loathe Medusa, but Evinrude is delayed when he is forced to take shelter from a flock of bats. The following morning, Medusa and Mr. Snoops send Penny down into a pirate's cave to find the gem, with Miss Bianca and Bernard hiding in her skirt pocket. Hubie and his friends manage to sneak into the cave when Snoops and Medusa weren't looking. The team soon find the Devil's Eye within a pirate skull; as Penny pries the mouth open with a sword, the mice push it out from within, but soon the oceanic tide rises and floods the cave. Miss Bianca, Penny, and Bernard barely manage to retrieve the diamond and escape. Hubie and his friends make it out by diving into the hole in which the water was coming from and out into the ocean, where they find the shore and reunite with Bernard and Miss Bianca.

Medusa plans to keep the diamond for herself, hiding it in Penny's teddy bear while holding Penny and Snoops at gunpoint. When she trips over a cable set as a trap by Bernard and Bianca, Medusa loses the bear to Bonkers who picks it up and calls Penny, who runs away with him. The local animals arrive at the riverboat and aid Bernard and Bianca by trapping Brutus and Nero, then setting off Mr. Snoops's fireworks to create more chaos. Meanwhile, Penny and the team commandeer Medusa's swamp-mobile, a makeshift airboat. Medusa unsuccessfully pursues them, using Brutus and Nero as water-skis, and is left clinging to the boat's smoke stacks as the irritated Brutus and Nero circle below.

Back in New York, the Rescue Aid Society watch a news report of how Penny found the Devil’s Eye, which has been given to the Smithsonian Institution, and how she has been adopted. The meeting is interrupted when Evinrude arrives with a call for help, sending Bernard, Bianca and Hubie and his friends, including Stanley on a new adventure.

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