If Cerebella kills an opponent with her Battle Butt attack, "2000" will appear on screen in a similar fashion as when killing an enemy in Battletoads. This was an intentional reference placed into the game by Mike Z.

One of Peacock's crouching attacks, "Curb Your Shoe", kicks the opponent with the Kuribo's Shoe from Super Mario Bros. 3

Filia's alternative color palettes reference several anime characters, such as Hatsune Miku from the VOCALOID series, Senjougahara Hitagi from animes "Bakemonogatari" and "Nisemonogatari", and Rei Miyamoto from Highschool of the Dead.

Ms. Fortune's winning quote, "Claws encounter of the furred kind!", is a reference to the 1993 video game Bubsy in Claws Encounter of the Furred Kind.

If Valentine loses by timeout, she explodes into a mass of bones as if hit by a Mortal Kombat 3 fatality,with multiple skulls and ribcages.

If Peacock uses the "Shadow of Impending Doom" special, there is a chance that she will drop a steamroller on the opponent, similar to what Dio does when using his stand, The World, in JoJo's Bizarre Adventure. Avery even makes a similar noise when attacking.

Fans of the Hasbro TV show "My Little Pony: Friendship is Magic" set out on making a fighting game called "My Little Pony: Fighting is Magic." Developed by a team of fans called "Mane6," the game would use locales and characters from the show. However, after receiving a cease and desist from Hasbro Corporation, Lab Zero Games added a stretch goal in their Skullgirls Indiegogo campaign to allow the Mane6 dev team to use the Skullgirls engine. The stretch goal was met, allowing Mane6 to continue their game with different assets.

According to Lab Zero Games, the total cost of adding a new character to the game's roster is more than $150,000:

Parasoul's level 3 blockbuster, Inferno Brigade, resembles one of BB Hood's supers from Marvel vs. Capcom 2 and Darkstalkers.

The stage "Little Innsmouth" is a reference to the H.P. Lovecraft novella, "The Shadow Over Innsmouth".

The cafe featured in Filia's story, MadMans Cafe, is a reference to the popular fighting game website of the same name. The artwork for the cafe is a parody of the Edward Hopper painting "Nighthawks".

According to one of the artists behind Skullgirls, Umbrella's dialogue at the end of Parasoul's story mode is a reference to Terry Bogard's catchphrase "C'mon! Get serious!" in Garou: Mark of the Wolves.

If Big Band wins a fight, he has a chance of saying "I have to go now. My planet needs me." This is a reference to The Simpsons episode "The Itchy & Scratchy & Poochie Show".

Parasoul's kid sister, Umbrella, was originally going to be included in the initial release of the game, but was dropped early on in favor of Valentine and Double to increase the number of combat-capable villain characters. Unused graphics and voice clips for Umbrella can still be found within the game's files, although Umbrella would later become a DLC character in the game's first Season Pass in 2021.

Fukua was originally meant as an April Fools joke poking fun at Capcom's announcement of Decapre, a clone character for Cammy, being included in Ultra Street Fighter IV. However, due to fan feedback, she got to stay in the game.

The scene in Fukua's reveal trailer and story mode where Filia is asleep in bed is a reference to the ending of Super Mario Bros. 2.

Inside the game's files is an unused graphic with an alternate spelling of Squigly's name. It appears that it was meant to be used on the character select screen.

The Cathedral stage has pictures of Venus and Aeon seen in the stained glass windows. Both characters have only been seen in Double's ending, and had a chance of being downloadable characters during the game's Indiegogo campaign, but they were not chosen.

Umbrella's design seems to be inspired by the mascot of the salt-producing company Morton Salt.

When using Cerebella's Lock & Load move, there's a 0.2% chance that a crudely-drawn cat will randomly appear.

This was derived from one of the animators drawing a cat in one of the frames but forgetting to take it out. Mike Z insisted that the cat should remain as an Easter egg, with the staff agreeing on the condition that Mike Z would animate it himself.