In 1912, 13-year-old Indiana Jones (or Indy) is horseback riding with his Boy Scout troop in Moab, Utah. He discovers a group of thugs who have found a golden cross that belonged to Coronado and steals it from them, hoping to donate it to a museum. The men give chase through a passing circus train and Indy escapes but the sheriff makes him return the cross. Meanwhile, Indy's oblivious father, Henry Jones, Sr. is logging extensive research on the Holy Grail in a diary.
In 1938, after recovering the cross (with help from Daring Do) and donating it to Marcus Brody's museum, Indy, Daring, 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, Rarity, Rainbow Dash, Fluttershy, Applejack, Pinkie Pie, Spike, Apple Bloom, Sweetie Belle, Scootaloo, Shining Armor, Princess Cadence, Zecora, Stephen and Stuingtion's Engines, Rattlesnake Jake, and Dusty Crophopper are introduced to Walter Donovan, who informs them that Henry has vanished while searching for the Grail, using an incomplete inscription as his guide. Indy then receives Henry's Grail diary via mail from Venice. Realizing that he would not have sent the diary unless he was in trouble, Indy, Daring, Thomas, Twi, and their friends, and Marcus travel to Venice, where they meet Henry's Austrian colleague, Dr. Elsa Schneider. Beneath the library where Henry was last seen, Indy, Daring, Thomas, Twi, and their friends, and Elsa discover the tomb of a First Crusade knight, which also contains a complete version of the inscription that Henry had used, this one revealing the location of the Grail. They flee, however, when the catacombs are set aflame by the Brotherhood of the Cruciform Sword, a secret society that protects the Grail from evildoers. After being chased in speedboats, Indy, Daring, Thomas, Twi, and their friends, and Elsa capture the Brotherhood's leader, Kazim, and Indy and Daring tell him that their goal is only to find his father and that they have no interest in finding the Grail. Kazim tells them that Henry was abducted to Castle Brunwald on the Austrian-German border. Marcus later reveals a map drawn by Henry of the route to the Grail, which begins in Alexandretta. The whole group sets off for Castle Brunwald, on the way T.C. tells the group more about his father and even tells them what happened to his mother.
At Castle Brunwald, Indy, Daring, Thomas, Twi, and their friends rescue Henry, but learn that Elsa and Donovan are actually Nazis using them to find the Grail for them. Meanwhile, Marcus is captured in Hatay, Turkey while waiting with Sallah for the Joneses, Daring, Thomas, Twi, and their friends. The Joneses, Daring, Thomas, Twi, and their friends also discover that the Nazis have also captured T.C.'s Father (General Dedrich) and Mr. Great White (Sharky's father) and then escape from Castle Brunwald and recover the diary from Elsa at a Nazi rally in Berlin, barely escaping an accidental face to face encounter with Adolf Hitler. They board a Zeppelin to leave Germany, but the Zeppelin soon turns around and the Joneses, Daring, Thomas, Twi, and their friends, General Dedrich, and Mr. Great White escape in a biplane and aboard the Gold Rust. They crash while engaging in a dogfight with the Luftwaffe, but manage to beat off the attacking planes from the ground.
The team meets up with Sallah in Hatay, where they learn of Marcus's abduction. The Nazis are already moving towards the Grail's location, using the map possessed by Marcus. In exchange for a Rolls-Royce Phantom II, the Sultan of Hatay has given the Nazis full access to his equipment for the expedition, including a large tank. Indy, Henry, Daring, Thomas, Twi, and their friends, General Dedrich, Mr. Great White, and Sallah find the Nazi expedition, which is suddenly ambushed by the Brotherhood. During the battle, Henry is captured while attempting to rescue Marcus from the tank. The younger Jones, Daring, Thomas, Twi, and their friends, General Dedrich, and Mr. Great White pursue the tank on horseback and, with the aid of Sallah, save Henry and Marcus before the tank goes over a cliff.
Indy, Henry, Daring, Thomas, Twi, and their friends, General Dedrich, Mr. Great White, Marcus, and Sallah catch up with the surviving Nazis, including Donovan and Elsa, who have found the temple where the Grail is kept but are unable to pass through the three protective booby traps. Donovan shoots Henry and Pinkie Pie, mortally wounding them, and Indy, Daring, and Rainbow forced to risk their lives in the traps to find the Grail and use its healing power to save Henry and Pinkie Pie. Using the information in the diary and followed by Donovan and Elsa, Indy, Daring, and Rainbow successfully reach the Grail's chamber, which is guarded by the last knight. He has been kept alive for seven hundred years by the power of the Grail, which is hidden among dozens of false Grails. Elsa betrays Donovan by deliberately giving him a false Grail, which causes him to decay into dust upon drinking from it. Indy and Daring then uses their expert knowledge to find the true Grail, which the knight warns cannot be taken beyond the great seal at the temple's entrance. Indy fills the Grail with water and takes it to Henry, who is instantly healed and then Daring and Rainbow give the rest to Pinkie Pie and she gets healed. Elsa then takes the Grail past the great seal, forgetting the knight's warning. The temple begins to rapidly collapse and, even as Indy attempts to save her, Elsa falls to her death into an abyss. Indy nearly suffers the same fate, but is saved by Henry. (And while no one's looking, Steam Mech retrieves the Grail) The Joneses, Daring, Thomas, Twi, and their friends, General Dedrich, and Mr. Great White, Marcus, and Sallah then narrowly escape the collapsing temple, and the team rides out of the canyon and into the sunset.
- Daring Do, Zecora, Stephen, Steamy, Puffy, Evan, Shai-Shay, Sharky, Willy (the train), Mako, Rattlesnake Jake, The Miner Trains (Steam Grindor, Steam Mech, Mucker, Steam Claw D., Steam Driller, Steam Sweeper and Steam Exavator), T.C., Hugs, Buzz, J.J., Socky, Skunky, 1206, The Planet Trains (Zip, Dazzlen, Dusten, Gustis, Rings, Uray, Tune and Shiver), and Dusty Crophopper will guest star in this film.
- T.C.'s Father (General Dedrich) and Mr. Great White will make a special apperence in this film.
- The storyline concludes in Thomas and Twilight Sparkle's Adventures with Indiana Jones and the Kingdom of the Crystal Skull.