If the game detects that the game is pirated, a pitch-shifted and reversed version of the main theme song will play on the title screen. When this song is plugged into a spectrogram, the end of the song will form The Devil in the 3 forms of his boss fight - Regular, skeletal and giant face.

Found within the game's files is an unused title theme for Cuphead with lyrics. This may have been composed during early stages of the game's development, as the lyrics describe a completely different plot from what was featured in the final game.

According to Jake Clark, when he was designing Cala Maria, The character went through a notable amount of changes before settling with the final design.

Before being settled for having a octopus on her head. Her face originally had fish fins on the side of her face, she had various different types of crowns(along with different hair styles) a starfish was considered to be used at one point, she had a round earring on her nose, a crab tattoo on her right arm, and wearing gloves.

It was also considered for her to hold a weapon(A sword and an anchor). As well the following for her attacks. Cala using her tail, summoning swordfish, a giant crab, and using a title wave.

In the games files, you can find an unused boss based off of a Pachinko machine that goes by the name "Pachi-Pachi".

The developers planned to surpass the Guinness World Record for number of boss battles in a run and gun game by having over 30 to the record's 25.

According to animator Jake Clark (who animated most of the bosses in the game), the mermaid boss was inspired by not only Betty Boop, but also Disney's Ariel (for the bra) and the moth character from the Disney Silly Symphony cartoon "The Moth and The Flame".

In the background of the garden level where the player has to fight the potato and carrot bosses, there's a bag that says 'Acme Grow'. This is a reference to classic Warner Bros. cartoons, which would often contain fictional "Acme" products.