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

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.

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 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.

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.

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.

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.

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".

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

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

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.

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.

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.

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.

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

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 Leviathan's possible quotes during Squigly's Daisy Pusher attack is "For I beheld Satan as he fell from Heaven like lightning!" This is a reference to one of Raul Julia's lines as M. Bison from the 1994 live action Street Fighter adaptation. This line was also originally used in Luke chapter 10, verse 18 in the Holy Bible.

During the 2013 Skullgirls Evo Breast Cancer Donation Drive, anybody who donated $50 USD could request a personal voice message from the voice actors of the game. One of the requests was for Peacock's voice actress, Sarah Anne Williams, to sing the song "Yakko's World" from the popular 90's cartoon Animaniacs.

Eliza is based and named after 2 people: Elizabeth Bathory, a Hungarian Countess and serial killer who believed that by using her victim's blood she would remain beautiful, mirrored by Eliza's own use of blood, and Elizabeth Taylor, the actress who played the title character in the 1963 film, Cleopatra.

When Filia beats Robo Fortune she will say "does she dream of electric mice?", a reference to the science fiction novel "Do Androids Dream Of Electric Sheep?" By Phillip K. Dick.