Rather than attending the Muppets' annual Halloween party, Gonzo and Pepe are en route to participate in a fear challenge event in the mansion of Gonzo's favorite magician, The Great MacGuffin. Upon being dropped off by their driver (Yvette Nicole Brown), Gonzo and Pepe meet the caretaker (Darren Criss), along with several ghosts and singing busts outside the mansion who perform "Rest In Peace."
The duo is then greeted by their "Ghost Host" (Will Arnett) who informs them that Gonzo was invited there to see if he can survive one night inside this haunted mansion. And if they cannot, they will be trapped there forever. While Gonzo claims that he is afraid of nothing, Pepe is scared by everything he sees. The two explore the mansion and, after meeting Madame Pigota, Gonzo and Pepe come across a room full of several ghosts resembling their Muppet friends who put on a vaudevillian-esque show entitled The Mansion Show (featuring At the Dance style jokes, the stand up comedy of Gauzey the Hatbox Bear, and a performance of "Life Hereafter"). The host reappears and informs Gonzo that in order to survive his time in the mansion he must face his fears in Room 999.
As Gonzo is led off to the room, Pepe meets and becomes entranced by the mansion's resident bride Constance Hatchaway (Taraji P. Henson). It is revealed via song in the "Tie the Knot Tango" that she plans to marry Pepe and then, as explained by the ghosts of her past husbands, kill him.
Gonzo enters Room 999 and faces his biggest fear: that no one will like him if he does not continue to do dangerous stunts. Gonzo starts to turn old as his reflection taunts him, but recalling a conversation with Kermit that he had earlier, Gonzo realizes that he can be great by just being Gonzo and, upon looking out at the party he's missing, he realizes that his bigger fear is losing his friends (and no stunt is worth that). Gonzo rejects the allure of the mansion and its challenge and is ready to leave to reunite with his friends.
The host appears to congratulate Gonzo for facing his fears and tells him that he is free to go. But upon realizing that sunrise is approaching and that Pepe is in trouble, Gonzo refuses to leave the mansion without him. With the help of the host's candelabra, Gonzo is able to find Pepe and stop the wedding. The pair jump out the window and make it out of the mansion just as the sun rises. The host meets them outside, revealing himself to be the ghost of the Great MacGuffin, and is impressed that Gonzo was willing to do what he could not - face his fears. The pair are picked up by their driver and head off to meet the rest of their friends for a post-Halloween breakfast, joined by the Hitchhiking Ghosts as "Dancing in the Moonlight" plays on the car's radio and a rainbow appears in the sky.
- The Great Gonzo as himself
- Kermit the Frog as himself and a ghost
- Miss Piggy as herself and Madame Pigota
- Fozzie Bear as Gauzey the Hatbox Bear
- Pepe the King Prawn as himself
- Chip as Pickwick
- Rowlf the Dog as the Organist
- Robin the Frog as the Birthday Ghost
- Sam Eagle and Pops as the Duelists
- Crazy Harry as Alexander Nitrokoff
- Janice as Tightrope Girl and a member of the Phantom Six
- Dr. Teeth and the Electric Mayhem as the Phantom Six
- Yolanda the Rat as Medusa
- Mahna Mahna and the Snowths as the Gargoyles
- The Muppet Newsman as the Aging Man
- Annie Sue and Gaffer as the Werecat Lady
- Dr. Phil Van Neuter as the Black Prince
- Bunsen and Beaker as Staring Busts
- Link Hogthrob, Dr. Julius Strangepork, and Miss Poogy as the Quicksand Men
- Wayne and Wanda, Beverly Plume, Howard Tubman, Miss Cartier and Beautiful Day Monster as Ballroom Dancers
- Assorted Happy Haunts: Scooter, Clean Gene, Big Mean Carl, Mo Frackle, J. G., Statler and Waldorf, Bobo the Bear, The Swedish Chef, Marvin Suggs, Nigel (Muppet Show), Nigel (Muppets Tonight), Joe from Legal, Pokey, Mulch, Dr. Phil van Neuter, Squid Ghost, Chickens, Frackles, Skulls, Ghosts
- Constance's Husbands
- Caretaker's Dog
- Mummy and Duddy
- Uncle Deadly as the Minister of Constance's Wedding
- Andy and Randy Pig as the Grady Twins from The Shining
- Will Arnett as the Ghost Host/The Great MacGuffin
- Darren Criss as the Caretaker
- Taraji P. Henson as Constance Hatchaway
- Singing Busts
- Geoff Keighley as Uncle Theodore
- Pat Sajak
- Craig Robinson
- Skai Jackson
- Justina Machado
- Chrissy Metz as Harriet the Opera Singer
- Danny Trejo as Huet
- Sasheer Zamata as Mary
- Alfonso Ribeiro as Fred
- Jeannie Mai as Maude
- Ed Asner as Claude 
- John Stamos
- Yvette Nichole Brown as a Hearse Driver
- Kim Irvine as a Maid
- The announcement of the film came with a series of YouTube videos hosted by the Muppet, "Uncle Deadly" which went over the histories of the Haunted Mansion, Phantom Manor and Mystic Manor.
- Many of the ghostly Muppet costumes are recycled from The Muppet Christmas Carol.
- The Mansion's address number is 924, a reference to Jim Henson's birth date.