One of the enemies in Pizza Tower, the Pickle, is based on the Roper enemy from the game Memoirs of Magic, with modifications to more closely resemble a pickle as to fit the game's food theme. On the "Oh, Shit!" level, graffiti can be seen of a Pickle with a muscular arm bearing a tattoo reading "MOM", which has a double-meaning as both a reference to a stereotypical tattoo and an acronym for its origin game's name.

When one defeats Spoiler:The Doise, The Noise's exclusive boss fight, he is Spoiler:killed by what appears to be a fake version of Peddito. If one rematches the Doise on the hub world, The Noise will simply find his bloody corpse and look on in confusion. Fitting with this joke, in the final boss, the Doise does not have a phase in the boss rush, instead just having his corpse thrown on the ground by Pizzaface, giving the player some time to breathe, then disappearing.

Two of Peppino's sprite animations are references to non-verbal Italian gestures:
• One of his idle animations has him bite his hands, a threatening (and sometimes sexually loaded) gesture known as "si t’ancagliu" or "when I catch you, I'll kill you".
• The pose he does on the P-rank screen is "l'ombrello" or "the umbrella", an Italian equivalent to the middle finger.

Pizza Tower shifted from a puzzle/exploration-based game to a fast-paced game during development in response to videos of people speedrunning the SAGE 2019 demo.

Before the Pizza Priest and Stupid Rat were created, some transformations had unique objects associated with them, two that would've taken a similar role to the priest are in the game's files:
• A tombstone, which would turn Peppino back from a ghost
• A pile of corn, which would distract Mort and free Peppino from him, referencing how corn was used as a health pick-up in Mort the Chicken
and some blocks that could only be broken by transformations, like the rat, also remain in the files
• A barrel block
• A hot metal block, which would be destroyed by both knight and rocket transformations
• A ball block
• A Gustavo and Brick block

When replaying through an already-completed save file, one can change the Peppino's Final Judgement ranking screen. This was not always the case, as originally attempting to change the judgement would show a screen of an ominous Peppino, drawn with details than his sprites but less detailed than his battle introduction portraits, with the caption "THE JUDGEMENT WAS FINAL. PLEASE TRY A NEW SAVE FILE." This screen would eventually be implemented in the Halloween update, albeit without its text, when one attempts to exit a newly introduced bonus level through the ghost transformation.

The Noise, Pepperman, and The Vigilante were all planned to be playable at some point - Vigilante and Noise were both intended to be regular playable characters, while Pepperman was intended as a one-level only character on Refrigerator-Refrigerador-Freezerator, much like Gustavo & Brick. All 3 of them have code and animations left over in the game, of varying compatability. The Noise would later be re-added in a future update due to all of his animations being completed.

The Captain Goblin on the Crust Cove level originally was called Captain Bomb and had a more humanoid design. The only animations made for him are simply the same image rotated and modified.

Pizzahead's third phase was originally going to be, instead of a boss rush, Pizzahead growing desperate and starting to violently attack Peppino directly through kicks and punches rather than cartoon gags. This was scrapped as the development team didn't want Pizzahead to be perceived as an angry character as much as just an evil clown.

The Weenie and Burger Golfer enemies are based on Peter and Mac, the protagonists of an unmade Pizza Tower predecessor called Weenie Cop.

Many fans and critics have noted similarities between the aesthetic of Pizza Tower and "off-model" 1990s cartoons such as Ed, Edd, n' Eddy and The Ren & Stimpy Show. Game creator McPig has claimed not to be a fan of those series and to have not been influenced by them, although he did still take influence from the more cleanly-drawn SpongeBob SquarePants, and an unclear inspiration from 1990s cartoons would still be cited on the game's Steam store page description.

According to programmer Sertif in a reply to a now-deleted tweet, Gustavo's steed Brick the rat is intended to be female. Composer ClascyJitto corroborated this and assumed that the reason fans did not know this beforehand was because it was not overtly stated in the game.

"Meatophobia", the ominous background music that plays when approaching Pillar John and activating Pizza Time, features a timestretched version of the same hip-hop orchestral hit sample used in the Small Graffiti sound effect from Jet Set Radio.

Gnome Forest was originally supposed to have the player switch between Peppino and Gustavo & Brick constantly, with them being in a race to deliver more pizzas. This was scrapped in favour of a stage that almost exclusively features Gustavo & Brick so that players could more easily get used to their controls.

In the Slum area of the hub world, there is a peaceful green cheeseslime named Snotty. If one is to kill Snotty, a permanent tombstone will be placed in the hub world, but if he is kept alive, he will be added to the Crumbling Tower of Pizza stage as a rescue, and the save file with which the game was completed will get a stamp saying "Snotty Approved". Saving Snotty does not contribute to the game's completion rating, and if one kills Snotty after the save file is Snotty Approved, the stamp will not be taken away.

The Noise was designed to be an obvious parody of the 1980s Domino's Pizza claymation anti-mascot "The Noid". The game's developer McPig has claimed that if The Noise wasn't based on The Noid, the boss would've had a parody of Vocaloid Hatsune Miku instead, in reference to Domino's App feat. Hatsune Miku, an app that became a niche YouTube Poop meme in the early 2010s.

Before release, fans claimed to hear the phrase "Door stuck" in the music track "Peppino's Sauce Machine", hailing from a Counter-Strike video released in 2007. This would lead to a semi-official remix from composer Mr. Sauceman titled "Peppino's Door Problem", which mixes the "DOOR STUCK!" audio from the video into "Peppino's Sauce Problem".

During development of Pizza Tower, multiple demo builds were released. Among these was a build known as the "Peter Griffin Experience", built off of the 2018 "early test build". This demo replaced every single sprite of Peppino with a highly compressed edit of a stock image of Peter Griffin from Family Guy made to resemble Peppino, and replaced one of the game's musical tracks with a fan-made Family Guy remix.

After this build, the Peter Griffin "arms resting" pose would appear in some builds as a taunt, albeit as an actual sprite and not an edit. A YouTube video titled "family" would also be posted by developer McPig showcasing the taunt, accompanied by the first note of the Family Guy theme song, likely referencing a Family Guy meme video that plays the first note of the theme song and abruptly ends. This taunt was removed for unknown reasons in the final game.

During development of the game, Peppino was stated to be a war veteran who suffers from post-traumatic stress disorder. Following the release of the game, this character trait would be retconned by the game's creator McPig, who suggested that he probably merely imagined his participation in conflict.

Two background characters featured in the Pizzascare level, a pink-clothed Peppino and a dark-skinned blue-clothed heterochromatic Noise, are fan characters known as Peddito and the Doise created by a fan named Robbie1scool. The Doise would be included as The Noise's exclusive boss in the Noise Update.