The film begins in an abandoned river boat in Devil's Bayou, where orphan Penny drops a message in a bottle containing a plea for help into the river. The bottle is carried out to sea and washes up in New York, where it is recovered by the Rescue Aid Society. Thomas, Percy, James, Toby, Emily, Gordon, Henry, Edward, Bash, Dash, Ferdinand, Luke, Charlie, Hiro, Victor, Kevin, Skarloey, Rheneas, Sir Handel, Peter Sam, Rusty, Duncan, Twilight Sparkle, Applejack, Pinkie Pie, Fluttershy, Rainbow Dash, Rarity, Spike, Apple Bloom, Sweetie Belle, Scootaloo, Shining Armor, Princess Cadence, and the Hungarian representative, Miss Bianca, volunteers to accept the case and chooses the janitor Bernard as her co-agent. They visit Morningside Orphanage, where Penny lived, and meet an old cat named Rufus. He tells them about a wicked woman named Madame Medusa who once tried to lure Penny into her car and may have abducted Penny this time.
The mice and our heroes travel to Medusa's pawn shop. While there, Pinkie finds Willy's compass and assumes that Willy was sneaking through and while he was running away, he lost his compass but was corrected that it fell out of his cab while he was getting on a roof storage shelf where our heroes find him. After he hops on the ground, Fluttershy embraces him. Then Scootaloo asks what he's doing here, Willy replies that he saw them leaving the orphanage and follwed them there. Then Steamy chuffs up with Rattlesnake Jake, with the same reason. Willy then asks to join them on their quest which they accept. Then they discover that she and her partner Mr. Snoops are on a quest to find the world's largest diamond, the Devil's Eye. With the help of an albatross named Orville, and a dragonfly named Evinrude, the mice and our heroes follow Medusa and Mr. Snoops 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.
Thanks to Bianca and Rarity's perfume, the mice and the others attract the attention of Medusa's pet alligators, Brutus and Nero. After the struggle they start to give up but are encouraged by Willy and Steamy, Thomas, Twilight, the others, Bernard and Miss Bianca escape, and find Penny. The following morning, Medusa and Mr Snoops send Penny down into the cave to find the gem, unaware that Miss Bianca and Bernard are hiding in her skirt pocket [while Twilight uses an invisiblity spell. Down there, Rarity uses her gem scanning power to locate the diamond and soon finds the stone 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, Bernard, and Heroes barely manage to retrieve the diamond and Willy helps them escape (due to being a whale).
The greedy Medusa steals the diamond for herself and hides it in Penny's teddy bear. When she trips over a cable, Medusa loses the bear to Penny, who runs away with it. Medusa retaliates with gunfire, causing the mice and the others to flee until they are met by Brutus and Nero, her alligators. Bernard, Miss Bianca, and Rarity trick them into entering a cage-like elevator, trapping them. Then Jake returns fire with his gatling gun at Medusa, and then Rusty blows up her gun with a missile.
Two of the gang set off Snoops' fireworks, making the boat sink. Penny and the gang use Medusa's "swampmobile". Medusa is left clinged to the boat's smoke stacks with Brutus and Nero attacking below.
Back in New York, the Rescue Aid Society watch TV to hear that the Devil's Eye is given to the Smithsonian Institution and Penny is adopted by a new father and mother. Thomas, Twilight, the others, Bernard and Miss Bianca remain partners in the Rescue Aid Society's missions and soon after depart on Orville, accompanied by Evinrude, to a new rescue mission.
- Willy, Steamy and Rattlesnake Jake guest star in this film.
- When the team fly to Devil's Bayou, our heroes are carried by Cadance's magic due to them being too big to ride an albatross.
- The storyline continues in Thomas and Twilight Sparkle's Adventures of The Rescuers Down Under.