The tune has been featured in five episodes of The Simpsons:
In "The Otto Show", Homer Simpson sings the lyrics to himself while sitting in a parked car.
In ″Duffless″, when Homer and other people are watching (unseen) images of violent car crashes.
In "Team Homer" the music is featured as Lunchlady Doris narrates as Martin Prince and Lisa Simpson model the new colorless school uniforms.
In "Sunday, Cruddy Sunday", the tune is played when Homer waits for his car to be fixed (coincidentally, the night that the episode first aired, Julius Wechter died).
In "Natural Born Kissers" as Homer and Marge are going to buy a new motor for the fridge, the radio airs an ad on divorce, the music for which is "Spanish Flea".
The tune has also been featured in the Japanese animated series Kareshi Kanojo no Jijou, by studio Gainax, with its lyrics changed into a looping "paya pa pa". The song is also very popular in Norway, as the theme for a cooking-program, and has also been used in commercials.
In the fingerprint scene in Rosewood's home in Beverly Hills Cop II, Eddie Murphy (playing Axel Foley) and Judge Reinhold (playing Billy Rosewood) improvised the idea of humming the tune. When Taggart (John Ashton) asks what the tune is, Rosewood replies, "The Dating Game!"
In the Australian sketch comedy The Late Show (1992–1993), the final episode of the redubbed segment "The Olden Days" features an instrumental version of "Spanish Flea" playing over the scene in which Governor Frontbottom departs the mudfields.
In American Pie 2, after Michelle sticks a trumpet up Jim's butt, the head counselor at band camp plays the Spanish Flea. In the middle of the tune, he realizes the funny taste on the mouthpiece.
In the video game Montezuma's Revenge, the song was used on the game introduction of a widely available pre-release version. The song was removed from the final version due to technical constraints.
It is used during the "Mitch's World" segment on the American syndicated morning television show The Daily Buzz.