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.

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.

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.

The stage Gehenna is named after a place outside ancient Jerusalem where the worshippers of Baal had child sacrifices and was described as "filled with the blood of innocents".

One of Beowulf's blockbusters involves performing three uppercuts that each create an image of a howling wolf, which launches the opponent in mid air. After that, an image of a moon appears as he performs a body slam on the opponent. This is a reference to the infamous Three Wolf Moon T-shirt on Amazon.com.

In River King Casino, it's possible to see Yuzuriha from Under Night: In-Birth, Matt and Woolie from the web series "Two Best Friends Play", and two dogs dressed as Chun-Li and Guile from the Street Fighter series.

During the indiegogo campaign for Skullgirls Encore a reward was given to anyone who donated 1,000 dollars, the reward was to submit an NPC character to be included in the game.
Animator "Zone", who created Zone-tan and also worked on the game, was one of the "Patron Saint" backers. Zone-tan later appeared in the Glass Canopy stage.

The Diamond Deflector, a special move that Cerebella can do, is the only attack in the game that can reflect projectiles.

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.

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.

In Fukua's story mode, the boss fight with Filia will always be set to Nightmare difficulty no matter what difficulty is chosen.

The "Politician Double" voice pack has a few specific pop culture references, including:
•The popular internet meme, "MURICA!"
•"WE'LL DO IT LIVE!", from Bill O'Reilly's infamous breakdown.
•"GREAT SKULLGIRL OR GREATEST SKULLGIRL?" is a reference to Steven Colbert, who jokingly asked if George W Bush was a "Great President or Greatest President?"
•"YOUR LIFE WAS FAIR AND BALANCED!" is a reference to the infamous Fox News slogan.
•"I DID NOT HAVE PUGILISTIC RELATIONS WITH THAT WOMAN!" is a reference to Bill Clinton's response after being accused of having an affair.
•"FOOL ME TWICE...uh...um...WON'T GET FOOLED AGAIN!" is a reference to Bush Jr. failing to remember the saying in a similar fashion.
•"HELP CONTROL THE PET POPULATION! HAVE YOUR PETS SPAYED OR NEUTERED!" is a reference to The Price is Right, when the host would say this exact line.

The game contains a character select portrait for Marie, the games final boss, suggesting that there were plans for her to be a playable character. She was later included as a candidate in a DLC voting campaign during a 2013 Indiegogo fundraiser, but lost to Eliza and Beowulf.

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.

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.

Eliza's Story Mode text data commentary contains an ASCII picture based on fan art of Maya Amano from Persona 2. When questioned about it, programmer Jonathan Kim explained that many messages and pictures like that found in the game's code were the result of him venting off steam while working on the game, and that after the game had been datamined by fans, he was forced to remove the majority of those messages and pictures, with some exceptions like the Maya Amano fan art.

Valentine's design was officially changed on May 13th, 2014 via a patch update. The update changed all parts of her design that involve a red cross against a white background to be altered so the cross is colored pink instead. This was done in order to prevent legal action by the International Committee of the Red Cross.

On May 23, 2022, it was announced that Valentine was receiving another design update that would incorporate pink shurikens that replaces the original cross. Not only will this affect Valentine's original sprite animations for both the console/PC and mobile versions of the game, but also any story mode art as well as other art pieces included in the Digital Art Compendium.

During Fukua's reveal trailer, the character Brain Drain is deciding which of the candidates featured in the video will become DLC and among those candidates is a dog. The dog gets rejected for being too cute and Brain Drain says "come back when you learn how to manipulate sand or something".

This is a reference to Iggy, a character from the JoJo's Bizarre Adventure series. In JoJo, Iggy is notable for being a dog who has the ability to manipulate sand.

On April 23rd, 2015, an update was released that altered some frames of animation for Filia, Fukua, and Cerebella. This was done with the purpose of removing or heavily obscuring some panty shots, as the developers considered them to be unnecesary fanservice.

Additionally, two unused animations for Filia were also removed from the Digital Art Compendium. These animations include a time over animation where Samson tears apart the top of her shirt while she tries to cover herself up, and a taunt where Samson spanks Filia hard enough to make her fly a short distance and land on her knees.