A young and somewhat reclusive Shaggy Rogers befriends a talkative stray dog, which he affectionately names Scooby-Dooby-Doo. On Halloween night, Shaggy and Scooby cross paths with three aspiring detectives: Fred Jones, Daphne Blake, and Velma Dinkley. Their adventure leads them into a spooky haunted house, where they encounter a ghost. After capturing and unmasking it, they discover the ghost is merely the costumed thief Mr. Rigby. This thrilling experience inspires the group to create their detective organization, Mystery Inc..
Fast forward ten years, after countless cases solved and adventures shared, Mystery Inc. seeks to partner with businessman Simon Cowell as an investor. However, conflicts arise when Cowell refuses to work with the group due to the perceived lack of teamwork from Scooby and Shaggy, which originates from their powerful friendship. Feeling like outsiders, Scooby and Shaggy decide to leave Mystery Inc., which leaves the team feeling disheartened.
Later that evening, at a haunted bowling alley, the duo faces threats from small hostile robots known as the Rottens. Just when things look bleak, they are saved by Dee Dee Skyes, the heroic Blue Falcon, and his sidekick robot dog, Dynomutt. It soon comes to light that the Rottens are minions of the villainous Dick Dastardly, who is bent on capturing Scooby as part of a sinister plot involving three supernatural skulls linked to a monstrous three-headed ghost dog, Cerberus.
Meanwhile, Fred, Daphne, and Velma are investigating the same mystery and soon learn that Scooby and Shaggy are with Blue Falcon. They encounter Officer Jaffe, who is revealed to be Dastardly in disguise, and all are imprisoned aboard his airship. Thanks to an escape aided by one of the Rottens, they reach out to Dee Dee via Dynomutt, discovering that they are being taken to Messick Mountain, where the last skull is hidden.
As they unravel Dick Dastardly's evil scheme, they find out he intends to unlock the gates to the Underworld to rescue his dog, Muttley, who was trapped during an ill-fated attempt to seize treasures from the Underworld. Only Scooby-Doo has the ability to unlock the gates.
The adventure leads Scooby, Blue Falcon, Dynomutt, and Dee Dee to Messick Mountain, where they venture underground to an enchanting Mesozoic island. Here, the last skull is guarded by Captain Caveman. After a brief confrontation, Fred and Shaggy join the fray, only for Fred to be revealed as Dastardly in disguise, who seizes the last skull. The Rottens subsequently eject the real Fred, Daphne, and Velma from the airship, and Dastardly destroys the Falcon Fury.
Scooby is ultimately captured and taken to Athens along with the skull. Conflict erupts between Fred and Blue Falcon while Velma, Daphne, Dee Dee, and Dynomutt attempt to mediate, but Shaggy steps in, taking full responsibility for Scooby's abduction as jealousy has strained their bonds. After delivering an uplifting speech, he inspires everyone to collaborate and rebuild the Mystery Machine to chase after Dastardly.
Upon arriving in Athens, Dastardly uses the skulls to unveil the gates to the Underworld, intending to use Scooby to unleash them. Their efforts are thwarted by the arrival of Scooby's friends in the flying Mystery Machine, who are shot down by the Rottens just as Dastardly releases Cerberus; thankfully, Dynomutt has landed them safely. As chaos ensues with Cerberus rampaging, Scooby races back to aid his friends. Dastardly finally reunites with Muttley in the Underworld, escaping with some of Alexander the Great's treasures.
Together, Scooby, Shaggy, Dynomutt, Blue Falcon, and Dee Dee devise a plan to trap Cerberus, while Velma, Fred, and Daphne delve deeper into Scooby’s lineage and its connections to the Underworld. With assistance from the Rottens, they successfully recapture Cerberus. However, Shaggy heroically sacrifices himself to secure the gates, only for the gang to discover another exit, which Scooby opens, leading to Shaggy’s reunion with them.
In a surprising twist, the Rottens betray Dastardly and Muttley, leading to Shaggy unmasking Dastardly, who turns out to be Simon Cowell in disguise. As it unfurls that the real Simon Cowell was genuinely busy judging a talent show, Velma exposes Cowell, revealing him as the true architect of the villainous plot against Mystery Inc. Ultimately, Dick Dastardly and Muttley are apprehended by Blue Falcon, Dynomutt, and Dee Dee and taken into custody.
Back home in Venice Beach, Scooby and the crew unveil their upgraded Mystery Inc. headquarters, celebrating alongside Blue Falcon, Dynomutt, and Dee Dee, who present them with an enhanced Mystery Machine as they set off to tackle their next mystery together. In a closing montage during the credits, the Mystery Gang experiences a rise in popularity, meeting Dr. Benton Quest after leaving the Rottens in his care. Blue Falcon and Dynomutt establish the Falcon Force, welcoming new members like Captain Caveman and others, while Muttley orchestrates Dastardly’s dramatic escape from prison.
