The Rescuers begins when a young orphan named Penny throws off a message in a bottle from an old abandoned luxury steamship on which she is held a prisoner. The bottle washes up in America and is taken to the Rescue Aid Society, a UN-like group, located in New York City, consisting of representative mice from all over the world. The mice read the message inside and learn that Penny was kidnapped. The Hungarian representative, Miss Bianca, is upset and volunteers to save the girl. The Chair of the society agrees, but on the condition that she chooses a partner for her safety. Of all the eager male delegates, she chooses Bernard, the janitor, for whom she has a soft spot. The two set out to the orphanage where Penny lived and there meet a local cat, Rufus, who has secretly retired from hunting mice. He tells them that Penny was a close friend of his, who despaired of being adopted by any family; that he had attempted to re-assure her; and that she is assumed to have fled of her own accord. The only alternative is to the effect that she was stolen by a mysterious, ill-meaning woman called Madame Medusa.

The rescuers, Bernard and Miss Bianca find themselves losing their faith; "What can two little mice do?" asks Bernard.

The mice then travel to Madame Medusa's pawnshop. While they are searching for clues, they hear Madame talking to her assistant, Mr. Snoops, on the phone. She yells at him for being unable to find a thing called the "Devil's Eye" and is furious to find out that Penny was sending messages in bottles. Angrily, she tells Mr. Snoops that she will take "the next flight down to Devil's Bayou" and leaves in her car. Bernard and Miss Bianca try to climb into her suitcase, but are thrown from it by Madame Medusa's reckless driving.

They recruit the help of an albatross named Orville, who flies them to Devil's Bayou. There, they are greeted by two muskrats named Ellie Mae and Luke, and a dragonfly named Evinrude, who loathe Madame Medusa for causing havoc in their neighborhood. With Evinrude's help, the rescuers are able to reach the ship on which Penny is held. Eavesdropping on Madame Medusa and Mr. Snoops, they learn that the Devil's Eye is a diamond coveted by Madame Medusa, and that Penny was captured so as to provide the criminals with one who could enter the underground pirate's cave wherein the gem is kept and recover it. Shortly after entering, Miss Bianca and Bernard attract the attention of Madame Medusa's half-tame crocodiles, Brutus and Nero. The crocodiles, who serve Madame as guards to prevent or spoil Penny's escape, attempt to devour the mice. In the resulting chase/battle, the ship's pipe organ as well as some curtains and Madame Medusa's patience are all broken. Madame, who cannot tolerate mice, complicates the pandemonium by her panic, her summons of the incompetent Mr. Snoops, and her use of a large gun.

Bernard and Miss Bianca escape, later to visit a disconsolate Penny. Penny still maintains hope that she might have a family of her own, yet her resolve is waning. The arrival of two well-meaning mice, who speak of co-operation and faith (a concept made familiar to her by Rufus) raises her morale. Together, the three devise a plan, which is put into action on the following day.

Meanwhile, Ellie Mae has roused her neighbors to action. They wait only for Bernard's message, which Evinrude is expected to deliver; once it has arrived, they will attack the steamship and rescue Penny, . Evinrude in fact has orders to find and bring Ellie Mae and her friends; en route to do this, he is pursued by hungry bats. He takes refuge in an empty bottle, delaying his message.

Penny struggles to free the Devil's Eye diamond from a pirate's cranium. The producers carefully designed every shot of this scene to slowly raise the tension. ^[3]

Come morning, Madame Medusa and Mr. Snoops send Penny into the cave to find the Devil's Eye. With her are Miss Bianca and Bernard, concealed in a pocket. The cave itself was once a treasure-trove used by pirates to store their plunder, before the crew (presumably) fought among themselves and killed each other. After a brief search, the friends discover the Devil's Eye inside the skull of one of the pirates; the means of its concealment therein is not explained. As they are opening the skull in order to bring the gem out, the oceanic tide rises and floods the cave. Miss Bianca, Penny, and Bernard barely escape, but do so successfully in both survival and seizure of the diamond.

Madame Medusa, passionate and jealous in greed, takes possession of the diamond immediately. Rather than give Mr. Snoops a 50% share in it as she had evidently promised, she takes it entirely for her own, concealing it in Penny's teddy bear. When both Mr. Snoops and Penny protest, she threatens to kill them.

Evinrude, eluding the bats, arrives at Ellie Mae's house. There, the tired insect is given a drop of Luke's stimulating beverage, whereupon he gives the order to charge. The locals run in a body to the riverboat.

As Madame Medusa is backing away from Penny and Mr. Snoops, clutching the toy bear in which is the diamond, Bernard trips her up by means of a cord. She loses control of the diamond. Before she can recover it, Ellie Mae and her friends attack, punishing Madame Medusa relentlessly for disturbing their peace. Madame Medusa retaliates with gunfire, causing the rebels to flee; they are met by Brutus and Nero. Just as the crocodiles are poised to kill the smaller animals, Bernard and Miss Bianca trick them into entering a cage-like elevator, which is then closed upon them.

The locals, following Penny's plan, set off flares and fireworks (used by Mr. Snoops on Madame Medusa's orders) into the riverboat's living quarters, while Penny and the mice commandeer Madame Medusa's "swampmobile" (a type of motor-boat, used by Medusa to traverse the bayou). Madame attempts pursuit, but is thwarted. By the flares and firecrackers, the ship is caused to explode and sink. Madame Medusa is left clinging to one of its smokestacks, the diamond gone from her, while the crocodiles (whom she had abused in her desperation) attack her from below.

The Devil's Eye is given to the Smithsonian Institute, and Penny is adopted by a new father and mother. 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.