Steamboat Willie is the short film which brought Mickey Mouse to his glory he has today, so it's no surprise that it's been refeneced in several different media.
- Several central Disney video games have Steamboat Willie themed levels, including Mickey Mania, Kingdom Hearts II, and Epic Mickey. Plus, in Epic Mickey 2 The Powerof 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.
- 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 ships 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.
- The Simpsons episode, "Itchy & Scratchy: The Movie" features a short but almost perfect parody of the opening scene of Steamboat Willie entitled Steamboat Itchy.
- 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".
- In a 2008 movie of Futurama called The Beast With A Billion Backs, the opening is a parody.
- The beginning of series 2 of the TV series Alexei Sayle's Stuff shows a black and white animation entitled Steamboat Fatty.
- In Pokémon: Diamond and Pearl, one of the episodes "Steamboat Willies!", is a play on the title.