In the Australian Outback, a young boy named Cody rescues and befriends a rare golden eagle called Marahute, who shows him her nest and eggs. Later on, the boy falls in an animal trap set by Percival C. McLeach, a local wanted poacher. When McLeach finds one of the eagle's feathers on the boy's backpack, he is instantly overcome with excitement, for he knows that catching an eagle that size would make him rich. McLeach throws Cody's backpack to a pack of crocodiles in order to trick the Rangers into thinking that Cody was eaten, and kidnaps him in attempt to force him to tell about the whereabouts of Marahute.
A mouse, the bait in the trap, runs off to alert the Rescue Aid Society. The message is sent to the Rescue Aid Society headquarters in New York, and Bernard and Miss Bianca, the RAS' elite field agents, 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, Willy, Sharky, Mako, Steamy, Evan, Rattlesnake Jake, The Miner Trains, and J.J. are assigned to the mission, interrupting Bernard's attempt to propose marriage to Bianca. They go to find Orville the albatross who aided them previously, but instead find Wilbur, Orville's brother. Bernard and Bianca convince Wilbur to fly them to Australia to save Cody. While our heroes use a special hover-train designed by the Miner Trains. In Australia, they meet Jake, a kangaroo mouse who is the RAS' local regional operative. Jake falls in love with Bianca and starts flirting with her, much to Bernard's annoyance. He serves as their "tour guide" and protector in search of the boy. With Willy vowing to terminate McLeach for good much to Thomas, Twilight and their friends' confusion.
At the same time, Wilbur is immobilized when his spinal column is bent out of its natural shape, convincing Jake to send him to the hospital. As Wilbur refuses to undergo surgery and flees, his back is unintentionally straightened by the efforts of the mouse medical staff to prevent him escaping through a window. Cured, Wilbur departs in search of his friends. As they journel to McLeach's hidout, our heroes ask Willy why he's so determined to terminate McLeach. He reveals that 37 years ago, before he became an engine, his whole pod was killed by poachers with only a golden watch given by his father as memory, much to the team's shock. At McLeach's ranch, Cody has been thrown into a cage with several of McLeach's captured animals after refusing to give up Marahute's whereabouts. Cody tries to free himself and the animals, but is thwarted by Joanna, McLeach's pet goanna. Realizing that Marahute's eggs are Cody's weak spot, McLeach tricks Cody into thinking that someone else has shot Marahute, making Cody lead him to Marahute's nest.
Bernard, Bianca, Jake, and our Heroes knowing that Cody is in great danger, jump onto McLeach's Halftrack to follow him. At Marahute's nest, the three mice andour heroes try to warn Cody that he has been followed; just as they do, McLeach arrives and captures Marahute, along with Cody, Jake, Fluttershy, Rainbow Dash (Knowing the pegasi will make him even more richer if he sells them as pets), and Bianca. On McLeach's orders, Joanna tries to eat Marahute's eggs, but discovers that they are just egg-shaped stones. Frightened that McLeach might be angry with her, Joanna drops the stones into the water. When she leaves, Bernard and th rest of our Heroes crawl out of the nest with the hidden eggs, grateful that Joanna fell for the trick. Wilbur arrives at the nest, whereupon and they convince him to sit on the eagle's eggs, so that they can go after McLeach. McLeach takes Cody and Marahute to Crocodile Falls, where he ties Cody up and hangs him over a group of crocodiles and attempts to feed him to them. But Bernard, riding a type of wild pig called a "Razorback", which he had tamed using a horse whispering technique used by Jake on a snake earlier, (and our heroes using the hover train) follow and disable McLeach's vehicle.
Mucker then notices McLeach with a rifle (or shotgun) and releaizes that he's trying to shoot the rope holding Cody above the water. To save Cody, Willy fights back and draws fire at McLeach while Bernard tricks Joanna into crashing into McLeach, then Scootaloo sends both of them into the water by pushing McLeach's boot. The crocodiles then turn their attention from Cody to McLeach and Joanna, while behind them the damaged rope holding Cody breaks apart. Willy keeps his rifle on McLeach in case he tries to get back to shore, but is more than willingly prepared to kill him. McLeach fights and fends off the crocodiles, but while Joanna reaches the shoreline, McLeach is swept over the waterfall. Bernard, Willy, Mako, Thomas, Percy, Twilight, Spike, Pinkie, Rarity, dive into the water to save Cody, as everyone else follows on the shoreline, (the ponies on the hover-train) but fail. Jake, Bianca, Cadence, Sharky, Rattlesnake Jake, and the Miner Trains free Marahute, Rainbow Dash, and Fluttershy in time for them to save both Cody, Bernard, Willy, Mako, Thomas, Percy, Twilight, Spike, Pinkie, Rarity, Applejack, Apple Bloom, Sweetie Belle, Scootaloo, Shining Armor, the narrow gauge engines, Bash, Dash, Ferdinand, Victor, Kevin, Gordon, Henry, Edward, Hiro, James, Toby, Emily, Charlie, Steamy, Evan, and J.J. (Marahute catches Cody and Bernard, Cadance grabs the trains with her magic, and Rainbow and Fluttershy catch Spike, the CMC, Pinkie, Twilight, Shining Armor, Applejack, and Rarity, [with a cloth]) sparing them McLeach's fate while the hover-train pluments to the bottom of the falls and explodes. Bernard, desperate to prevent any further incidents, proposes to Bianca, who eagerly and happily accepts while Jake salutes him with a newfound respect. All of them depart for Cody's home. Wilbur, whom they have neglected to relieve of his task, incubates the eggs until they hatch, much to his dismay.
- Willy, Sharky, Mako, Steamy, Evan, Rattlesnake Jake, the Miner Trains (Steam Grindor, Steam Mech, Mucker, Steam Claw D., Steam Driller, Steam Sweeper, and Steam Exavator), and J.J. guest star in this film.
- Locomotive 131 and the Jules Verne Time Train make an apperence in this film as the hover train.
- It is revealed that Willy was once a regular whale and that his family was killed by poachers [explaining why he was so determined to kill McLeach].