Taking place in Burbank, California on October 16, 2023, when Walt Disney Animation Studios turned 100 years old, its employees are leaving the Roy E. Disney Animation Building as their usual work day is done and Burny Mattinson (played by himself) imagines about the walls of the studio coming alive after an intern talks with him about the great history it made throughout the century.
Inside the studio, after the doors close, all is quiet when suddenly, Mickey Mouse (voiced once again by Chris Diamantopoulos), as seen in a production cel picture for Mickey's Birthday Party, comes to life, and calls Tinker Bell. Mickey questions if all of the employees are gone, in which Tink replies yes with a nod. Excited, Mickey, with his girlfriend Minnie (voiced by Kaitlyn Robrock), jump out of the picture, and Minnie gets many characters from the studio's filmography with her signature "Yoo-Hoo!" signal, whether traditionally or computer animated. Tiana (voiced by Anika Noni Rose), Pinocchio (voiced by Griffen Campbell), Judy Hopps (voiced by Ginnifer Goodwin), Nick Wilde (voiced by Jason Bateman), and Louis are the first to respond to the signal. Mickey calls to Bambi, Flower, and Thumper (voiced by TBA) while the latter calls to Orville, Bernard, and Bianca. Minnie, alongside Rhino, Mittens, and Bolt, enlists Peter Pan (voiced by TBA) to gather everyone from upstairs and he complies and flies off with Wendy, John, and Michael Darling.
During that, several gags of the characters interacting with one another happen all over the studio:
- Sisu (voiced by TBA) runs happily down the hall, followed by Jim Hawkins riding on his Alponian Solar Cruiser, Maui (voiced by Dwayne Johnson) changes into his hawk form and flies into the hall while reminding Aladdin and Abu to meet in the lobby, and the former says he'll see them there while sliding down the railing with them ending up with the rock trolls, Vanellope von Schweetz (voiced by Sarah Silverman) and Dodger ride on the former's candy kart, Elliott takes Cody on a fun ride, Wayne and Lanny slide down on their ropes, and the Ocean drops Moana into the hall along with Flounder, who needed water.
- Moana (voiced by Auli'i Cravalho) searches for water for Flounder (voiced by TBA). She approaches Merlin (voiced by TBA) (who was at the Snuggly Duckling coffee area with his sugar bowl, the Mad Hatter, the March Hare, Lucille Krunklehorn, Mrs. Potts, Chip, and Cogsworth, who tells the others that they have no time for tea), and with his help, he casts a spell using the magic words, "Chapeaulius Bowlius" to summon water in the Mad Hatter's hat for Flounder to swim in, much to the former's pleasure, who then tells a passing Penguin Waiter with a plate of spaghetti and meatballs that "There's a carp in [his] cap."
- After seeing an angry Stromboli trying to get a snack from the studio's vending machine, Elsa (voiced by Idina Menzel) freezes Hans in his frame after Anna (voiced by Kristen Bell) asks her if she thinks that all of the villains will be showing up (most likely because she would be worried about seeing Hans again after he tried to kill them in their film).
- Donald Duck (voiced by TBA) steps into an elevator with Carl, Terk, Humphrey the Bear, Ranger J. Audubon Woodlore, and Mother Gothel, when Flash (voiced by TBA) calls from across the hall to hold the elevator as he slowly takes his time walking to them, much to Donald's annoyance.
- Olaf (voiced by Josh Gad) draws on an animator's office desk humming "Friend Like Me" while using a book copy of Disney Animation: The Illusion of Life to boost his height while the Robin Williams Lost Boy, Timon (voiced by Nathan Lane) and Pumbaa pass by, with the former telling him to shake a leg (in which Olaf replies that the leg won't make a difference because it's all in the wrist) when the Genie bursts out of the drawing, sending Olaf to pieces and he uses his magic to put him back together.
- While Prince John, Thomas O'Malley, Chicken Little, Milo Thatch, Happy, the Headless Horseman, and Gaston (voiced by TBA), who sings about himself while doing so, are freshening up in the men's room for the picture, the Cheshire Cat appears in front of his face laughing. Then Minnie opens the door while covering her eyes and tells them to move it.
- While Christopher Robin, Kanga, and Eeyore try to free Winnie the Pooh (voiced by Jim Cummings) from his picture, Antonio Madrigal speaks with Cinderella's bird friends, Meeko, Cri-kee, Pascal, and Pua about meeting in the lobby while stopping Joanna the Goanna from eating Jaq and Gus before getting spooked by an angry barking Pluto.
- While Macbadger, Ratty, Mole, Ryder, Honeymaren, Sven, and Kristoff (voiced by Jonathan Groff) walk by, Kristoff warns some Dalmatian puppies, who are watching "Night on Bald Mountain" on a TV, that they'll have nightmares when Chernabog pops out of the TV, scaring the puppies and they run away and, in the process, run over Scrooge McDuck, who had just came out from his picture, causing him to drop his money bags, which Robin Hood and Little John grab.
- At this point, Flash finally makes it into the elevator, much to the exasperation of the others, especially Donald who he thanked for holding the elevator. Baymax then squeezes into the elevator after he makes it, increasing Donald's frustration and resulting him to be thrown into a quacking fit when the fully-occupied elevator closes its doors.
- While Ursula is being followed and annoyed by a lovestruck Splat, Cinderella and Prince Charming follow down the stairs, where the prince is the one to drop his shoe, much to their humor. Max picks up the shoe and runs off with it. Charming calls for Eric while Cinderella cheers for Max.
- Mickey was amused by this when he sees Kaa leading Clarabelle Cow with his hypnosis. He tries snap him out of it, but Rapunzel (voiced by Mandy Moore) says she has it covered, hitting Kaa with her frying pan offscreen to save her.
Mickey flies down the lobby stairs amongst some of the other characters to the entrance on Dumbo while Toby Turtle, Bowler Hat Guy, and DOR-15 avoid them. After Raya tells Mickey that the coast is clear, everyone goes outside to take the group photo. During the outside gathering, Wreck-It Ralph (voiced by John C. Reilly) bumps in front of the door, but is okay while Scar (voiced by Jeremy Irons) comes out feeling annoyed by those he refers to as "idiots" around him while Mr. Toad flies around the area on the Magic Carpet. Meanwhile, Ichabod Crane ducks from a ladder being held by Goofy. After Jock and Trusty walk by, Flora turns the building's hat structure pink while Merryweather quickly changes it back to blue. Oswald the Lucky Rabbit, (in black and white), is the last to arrive for the photo as he positions himself next to Pacha and Kuzco (in llama form, voiced by David Spade), in which Mickey allows Oswald to do so. Oswald tips his ears to say thank you to Mickey, but Kuzco warns him to keep a distance.
Everything is all set as Rafiki hands Goofy an old fashioned camera. However, things backfire as Goofy tries to find the timer button for the camera, he accidentally falls off the ladder, breaking it, shorty after Iago timed it with a three second countdown. Stitch chews on a part of the camera, but Lilo drags him away. Grumpy expresses a sarcastic comment saying "Well, that was fun." and Eeyore states that they might try again next century. Hiro, Go-Go, Honey Lemon, Wasabi, and Fred along with Friar Tuck, the Big Bad Wolf, Professor Ludwig Von Drake, Arthur Pendragon, and King Leonidas, among many of the others, leave in disappointment as the group photo is cancelled and ruined. Mickey tries to assure everyone that everything will be fine, but soon loses hope.
Just then, as everyone begins moping back inside, Alan-a-Dale plays "When You Wish Upon a Star" on his lute, joined in by Scat Cat on his trumpet, Mirabel Madrigal on her accordion, and Hathi Jr. using his trunk as a trumpet as everyone, including Taffyta Muttonfudge and Johnny Appleseed, returns to listen. Then the singing starts with Belle and the Beast's duet as Lewis and Wilbur Robinson, the Queen of Hearts, Penny, Pecos Bill, and Widowmaker listen, much to Hades' annoyance. Quasimodo, Pocahontas, Ariel, Scuttle, Baloo, and Mowgli also join in singing along. As the background chorus sings, the other characters help undo Goofy's mistake with the magic brooms sweeping up his camera in a pile, Fix-It Felix Jr. fixing it with his magic hammer, Hercules putting the ladder back in position with his super strength, and the Fairy Godmother using her magic wand to lift Goofy up over the ladder while he places the camera on top of it, and sets the timer. As the trio of Snow White, Mulan, and Asha (voiced by Ariana DeBose), Winnie the Pooh, Jiminy Cricket, and the rest of the cast sing the last verse of the song and smile for the photo, Tinker Bell then uses her wand to cut from the scene to where the group photo on the wall is taken, and the film ends with the tagline, "To all who have imagined with us, laughed with us, and dreamed with us, Thank You."
At the end of the credits, a dedication to Mattinson appears that reads "For our pal Burny and his 70 years of legendary storytelling at Disney Animation."
