In 1947, cartoon characters, commonly called "toons", are living beings who act out cartoons in the same way that human actors make live-action production. Toons interact freely with humans and live in Toontown, an area near Hollywood, California. R. K. Maroon is the human owner of Maroon Cartoon Studios; Roger Rabbit is a fun-loving toon rabbit, one of Maroon's stars; Roger's wife Jessica Rabbit is a gorgeous toon woman; and Baby Herman is Roger's costar, a 50-year-old toon who looks like an infact. Marvin Acme is the pratical joke-loving owner of Toontown and the Acme Corparation. Yogi and the gang then arrive in Toontown.
Maroon hires private detective Eddie Valiant to investigate rumors that Jessica is having an extramarital affair. Eddie and his brother Teddy used to be friends of the toon community, but Eddie has hated them, and has been drinking heavily, since Teddy was killed by a toon a few years earlier. When Eddie shows Roger photographs of Jessica "cheating" on him by playing patty-cake with Acme, Roger becomes distraught and runs away. This makes him the prime suspect when Acme is found murdered the next day. At the crime scene, Eddie meets Judge Doom and his Toon Patrol of weasel henchmen along with Dick Dastardly and Muttley. Although toons are impervious to physical abuse, Doom has discovered that they can be killed by submerging them in a mixture of solvents he refers to as "Dip."
Baby Herman insists that Acme's will, which is missing, bequeaths Toontown to the toons. If the will is not found by midnight, Toontown will be sold to Cloverleaf Industries, which recently bought the Pacific Electric sysetm of trolley cars. One of Eddie's photos shows the will in Acme's pocket, proving Baby Herman's claim. After Roger and Yogi and the gang show up at his office professing his innocence, Eddie investigates the case with help from his girlfriend Dolores while hiding Roger from the Toon Patrol. Jessica tells Eddie that Maroon blackmailed her into compromising Acme, and Eddie learns that Maroon is selling his studio to Cloverleaf. Maroon explains to Eddie that Cloverleaf will not buy his studio unless they can also but Acme's gag making factory. His plan was to use the photos to blackmail Acme into selling. Before he can say more, he is killed by an unseen assassin and Eddie sees Jessica fleeing the scene. Thinking that she is the killer, Eddie pursues her into Toontown. When he finds her, she explains that Doom killed Maroon and Acme in an attempt to take over Toontown.
Yogi and the gang, Eddie, Jessica and Roger are captured by Dick Dastardly and Muttley, Doom and his weasels and held at the Acme Factory, where Doom reveals his plan. Since he owns Cloverleaf and Acme's will has yet to turn up, he will take control of Toontown and destroy it with a mobile Dip-sprayer to make room for a freeway, then force people to use it by dismantling the trolley fleet and make a fortune through a series of businesses built to appeal to the motorists. With Yogi and the gang, Roger and Jessica tied up, Eddie performs a vaudeville act that makes all but one of the weasels literally die of laughter (the leader, Smart Ass, is subjected to the Dip) and confronts Doom. Doom survives being run over by a steamroller, revealing that he himself is a toon, and admits that he killed Teddy. Eddie eventaully dissolves Doom in the Dip by opening the drain on the Dip machine. As toons and the police arrive, Eddie discovers that an apparently blank piece of paper on which Roger wrote a love poem to Jessica is actually Acme's will, written in disappearing/reappearing ink. Eddie kisses Roger - proving that he has regained his sense of humour - and the toons and Yogi and his gang celebrate their victory. Then Ricochet Rabbit and Droop-a-Long join the gang in the end.
- Ricochet Rabbit and Droop-a-Long could be in it so they could join Yogi's gang.
- Dick Dastardly and Muttley will work for Judge Doom.
- This will be in honour of the film's 25th anniversary.
- Ricochet Rabbit will reveal to be Roger Rabbit's cousin.
- This will be the first 'Yogi Bear's Adventures' film to feature live-action characters.
- This film will be dedicated in memory of Bob Hoskins who died from pneumonia on April 29, 2014.