In the heart of Devil's Bayou, Louisiana, a young 6-year-old orphan named Penny drops a heartfelt message in a bottle, pleading for assistance, into the river. This bottle floats all the way to New York City, where it is discovered by the Rescue Aid Society, an esteemed international organization of mice operating within the United Nations. Among them, the enthusiastic Miss Bianca volunteers for the mission, enlisting the help of the timid janitor Bernard as her partner. Their quest takes them to Morningside Orphanage, where they meet Rufus, an elderly cat, who warns them about the wicked Madame Medusa, a woman with a dubious agenda who once tried to entice Penny.
Determined to uncover more, Bianca and Bernard investigate Medusa's pawn shop, where they learn that Medusa and her accomplice, Mr. Snoops, are after the legendary Devil's Eye, the largest diamond in the world. The duo soon discovers that the devious pair has kidnapped Penny and taken her back to Devil's Bayou, where they harbor evil plans involving two fierce crocodiles, Brutus and Nero. Mingling with a colorful cast of characters, including a friendly albatross named Orville and a quirky dragonfly named Evinrude, the brave mice set out to rescue Penny.
Upon reaching the bayou, they uncover Medusa’s dastardly scheme: she aims to force Penny into a treacherous blowhole leading to a pirate cave hidden beneath the waters, where the Devil's Eye awaits. With a flicker of hope, Bernard and Bianca devise a daring escape plan. They attempt to rally the local animals to assist them, but Evinrude faces unexpected delays due to a swarm of bats obstructing his path.
The following day, Penny is sent into the perilous cave by Medusa and Snoops, unaware that her new mouse friends are cleverly hidden in her dress pocket. A thrilling treasure hunt ensues as they locate the coveted diamond ensconced within a pirate skull. Just as Penny pries the treasure free using a sword, disaster strikes—an ocean tidal wave begins to flood the cave, forcing them to escape just in time with the diamond in tow.
However, Medusa, manipulative and treacherous, betrays Snoops and stashes the diamond in Penny's teddy bear as she threatens them at gunpoint. In a twist of fate, she falls victim to her own traps set by Bernard and Bianca, losing both the bear and the diamond to the nimble Penny, who makes a break for it. The local animals rally to support Bernard and Bianca, working together to outsmart Brutus and Nero, and igniting a chaotic display of fireworks that further complicates Medusa's pursuit.
In the climax of their exhilarating chase, Penny and the mice commandeer Medusa's swamp-mobile to make their escape, while Medusa, using the crocs as water-skis, finds herself precariously clinging to the sinking boat amidst havoc. After a series of misfortunes, Medusa's reign ends as she is left stranded, while Snoops selfishly profits from his escape on a raft.
Back in bustling New York, the Rescue Aid Society celebrates as they watch Penny's story unfold on the news, revealing how she bravely uncovered the Devil's Eye, which has been generously donated to the Smithsonian Institution. It is joyfully mentioned that she has also found a new family to adopt her. Just as they bask in the triumph, Evinrude rushes in with another urgent plea for help, catapulting Bernard and Bianca into yet another thrilling adventure.
