In 1957 during the Cold War, World War II veteran Indiana Jones, Daring Do, and his partner George "Mac" McHale are kidnapped by Soviet agents under Colonel Dr. Irina Spalko. The Soviets infiltrate a warehouse labeled "Warehouse 51" in Nevada and force Jones to locate a non-human corpse including a crystal skull, recovered ten years earlier. Upon its discovery, Mac reveals he is a double agent working for the Soviets, and Jones and Daring escape on a rocket sled. Jones and Daring, still pursued, arrives in the model town at the Nevada Test Site, minutes before an atomic bomb test, and takes shelter in a lead-lined refrigerator. Jones and Daring are arrested by FBI agents, who suspect him of working for the Soviets; and though freed (also bailed out by 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), is put on indefinite leave of absence from Marshall College.
Jones, Daring, Thomas, Twi, and their friends, are approached by greaser Henry "Mutt" Williams, who tells them that Harold Oxley had found a crystal skull in Peru, suffered a mental breakdown, and was later kidnapped. In return, Jones and Daring tell Mutt about the legend of crystal skulls found in Akator. Mutt gives Jones a letter from his mother, who is also held captive, containing a riddle written by Oxley in an ancient Native American language. KGB agents try to take the letter, but Jones, Daring, Thomas, Twi, and their friends, and Mutt evade them and reach Peru. At the local psychiatric hospital, Oxley's scribbles on the walls of his cell lead them to the grave of Francisco de Orellana, a Conquistador searching for Akator. They discover the skull at the grave, with Jones reasoning that Oxley had hidden it there.
Shortly afterward, Jones, Daring, Thomas, Twi, and their friends, and Mutt are captured by the Soviets and taken to their camp, where they find Oxley and Mutt's mother, Marion Ravenwood, who later reveals that Mutt is Jones' son, Henry Jones III. In the following struggle, Mac claims to Jones he is really a CIA double-agent to regain Jones' trust. Spalko believes that the crystal skull belongs to an inter-dimensional alien life form and holds great psychic power, and that returning the skull to Akator will grant the Soviets the advantage of psychic warfare. Oxley's mental conditions having devolved, Spalko uses the skull on Jones to identify a route to Akator. Jones and his allies escape with the skull into the Amazon, elude giant ants, and survive three waterfalls in a duck boat, where after Jones, Daring, and Oxley identify markings that lead them to Akator, unaware that Mac has been dropping transceivers to allow the Soviets to track them.
Having escaped a native tribe guarding the city and gained access to the temple, filled with artifacts from several ancient civilizations, Jones and Daring believe the aliens were "archaeologists" studying the different cultures of Earth. The team enters a chamber containing the crystal skeletons of thirteen enthroned inter-dimensional beings, with one missing its skull. Spalko places the captive skull on this skeleton, whereupon the aliens offer a reward in an language that Oxley deciphers. Spalko demands knowledge equal to the aliens' but dies during the transference thereof, while the aliens activate a portal to their own world. Jones, Daring, Thomas, Twi, and their friends, Marion, Mutt, and Oxley escape, while Mac and the Soviets are drawn into the portal. The survivors watch as the temple walls crumble, revealing a flying saucer rising from the debris, which vanishes into the "space between spaces" while the void left by its departure is flooded by the Amazon waters.
Sometime later, Jones is reinstated at Marshall College and made an associate dean. He and Marion are then married in church. Daring decides to give Rainbow Dash a chance to join her on her next adventures which the rainbow pegasus accepts.
- 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.