After finishing the story mode of a pirated copy of the game, a notice will pop up saying "What is the square root of a fish? Now I'm sad." This was most likely added to confuse pirates.

Skullgirls has a Guinness world record for most frames of animation per character. It has 11,515 frames of animation for its 8 playable characters (DLC characters not included) which is an average of 1,439 frames per fighter.

The game was originally planned to have alternate endings for Story mode for most of the cast except Double. There is unused text of these alternate endings complete with dialogue for what appear to be choices the player would make.

Filia: Would wish for her memories back despite Samson's protests

Cerebella: Asks the Skull Heart to make Vitale Mecidi love her and only her. When she returns home, however, Vitale has gone mad and murdered everyone present except Feng just so he can have Cerebella all to himself. Feng kills Vitale only for him to get back up, turned into a monster. Cerebella and Feng flee to the country, hoping to start a new life.

Peacock: She wastes time making inane wishes that the Skull Heart could not possibly grant, much to the Heart's annoyance.

Parasoul: Seemingly kills Umbrella to prevent another Skullgirl from being made. Umbrella reassures her with her dying breath that she did the right thing.

Ms. Fortune: Goes ahead with her original plan to revive the Fishbone Gang. They are revived as mindless servants of the now Skullgirl Fortune.

Painwheel: Wishes for "Freedom". The Skull Heart frees her from human emotion and restraint, making her into an incredibly powerful Skullgirl.

Valentine: Seals the Skull Heart away to prevent it being used again knowing that it can't be destroyed so easily. However, when she faked a deception against Brain Drain, she killed a character named Christmas, who was close to Brain Drain. For the sake of revenge, Brain Drain forces a control mechanism on Valentine and turns her into the same abomination as Painwheel. As this happens, the Skull Heart mocks Valentine's efforts.

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.

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.