Disney Media
- Several central Disney video games have Steamboat Willie themed levels, including Mickey Mania, Kingdom Hearts II, Epic Mickey, Phineas and Ferb: Across the 2nd Dimension, and Disney Speedstorm. Plus, in Epic Mickey 2: The Power of Two, a Steamboat Willie outfit can be obtained for Mickey.
- In the Mickey Mouse cartoon, April Fools' Day, Mickey and Mortimer get sent to the President's office to claim a million dollars. But Mortimer pretends to be Mickey and he is shown acting in Steamboat Willie.
- In the Goofy cartoon How to Be a Waiter from 1999, Goofy is shown an example of a movie, and Steamboat Willie is shown. But in that short, Willie is a shortened version titled Steamboat Goofy. After Goofy recreates the iconic opening, he crashes into Mickey's steamboat and sinks. Mickey even squawks at him for interrupting his own short.
- The iconic opening scene was parodied towards the end of Aladdin and the King of Thieves. Genie, having been swallowed by the giant turtle which carried the Vanishing Isle upon its back, came back out of the turtle's mouth in the steamboat from this film and was even in Mickey's form, whistling "Turkey in the Straw".
- In Runaway Brain, a still of the short appears in Mickey's wallet, with Mickey saying that it's an old photo.
- Since Meet the Robinsons in 2007, the scene of Mickey at the ship’s wheel whistling "Steamboat Bill" has been used for Walt Disney Animation Studios' production logo at the beginnings of the feature films. A modification was used for Tangled to mark that film as the 50th in their Classics line, the text saying "Walt Disney Animation Studios: 50th Animated Motion Picture" with the Mickey scene in the "0". An 8-bit version of the logo was used for Wreck-It Ralph, and in Frozen and Moana, Mickey's whistling was muted to allow their respective opening themes to play out over the logo.
- Italian weekly Disney comic Topolino has Donald Duck being at the ship's wheel in a June 2014 cover.
Non-Disney Media
Anime & Manga- In Pokémon the Series: Diamond and Pearl, the episode "Steamboat Willies!" is a play on the title.
Film
- In Saving Private Ryan, a German POW tries to win the sympathy of his American captors by mentioning Steamboat Willie, even mimicking the sound of the boat whistle from the film. Bonus points for the unnamed character appearing in the credits as "Steamboat Willie".
Television
- The beginning of series 2 of the TV series Alexei Sayle's Stuff shows a black and white animation entitled Steamboat Fatty.
Video Games
- In Infinite Craft, "Steamboat Willie" is a possible crafting recipe.
Web Video
- Super Eyepatch Wolf's video Why Cuphead Looks So God Damn Good
includes a short clip of Mickey whistling at the wheel when talking about the era of stylization that inspired the game's visual design.
Western Animation
- Animaniacs (2020): On the segment The Warner's Vault from episode 25 features Yakko Warner parodying the opening sequence of the cartoon.
- In the first episode of Duckman, he finds home movies of his life that parody various cartoons. The first is this one, in which Duckboy demands to be captain and screams at a polite mouse to relinquish control. Another character (patterned after the Wise Little Hen) then appears to remind Duckboy that he'll have a much more rewarding and successful life if he behaves like the mouse. After stopping the clip, Duckman grouses about "the squeaky-voiced rodent" making money off being nice.
- In the Futurama movie titled The Beast with a Billion Backs, the opening is a parody of Steamboat Willie.
- The Simpsons: "Itchy & Scratchy: The Movie" features a short but almost perfect parody of the opening scene of Steamboat Willie entitled Steamboat Itchy.
- The South Park episode "The New Terrance and Philip Movie Trailer" has Russell Crowe's Fightin' 'Round the World, where Russell Crowe would go around on his tugboat looking for a scrap. One such trip had Crowe assume a similar posture while spinning the helm the same way Mickey Mouse did.
- Uncle Grandpa: In the episode "The Fan", Nubert Nimbo who is a big fan of Uncle Grandpa, shows to him one of the episodes of Uncle Grandpa's show. The episode in specific is a scene where Uncle Grandpa sings while driving a steamboat, having also a similar design used by the original animation.
