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.

The title card for the "Gnome Forest" level became the subject of the "Lario" meme, which features an edited version of the card with the word "Lario" above the characters' heads as a mash-up of "Sneaky Snitch" by Kevin MacLeod and "Superstar" by Young Thug. This meme would go on to be referenced within Pizza Tower itself, with Gnome Forest's secret music being renamed "Everybody Wants To Be A Secret" after the meme became popular.

If the player idles on the main menu for about 40 seconds without inputting anything, Peppino will eventually jumpscare them, causing the game to close itself.

One of the Pizza Grannies on the hub world claims that the player can type "/pizza" to order a pizza from Peppino's Pizzeria as an "Instant Pizza Gaming Promotion". This is a reference to a promotion done for EverQuest II, where the player could type "/pizza" into the game chat to open up the Pizza Hut website.

The game was originally planned to feature a "Heat Meter" that kept track of how many enemies the player was able to defeat in a row. More consecutive kills would build up the player's "heat," while getting hit and spending too much time without attacking enemies would lose heat. Higher heat levels would additionally make enemies more aggressive, increase the amount of points lost from taking damage, and grant Peppino new attacks. The development team compared this mechanic to a similar one found in God Hand.

The Heat Meter was fully implemented in a demo released to Patreon backers in April of 2021, but was simplified in later builds before ultimately being removed in October of 2022 due to mixed audience responses; several animations associated with it were also reconfigured for other purposes. Despite this, graphics for the Heat Meter and code for the more aggressive enemy behavior are still present in the final game. The Pizza Slug, Shrimp Thug, and Tribe Cheese enemies would also have their high-aggression modes enabled by default.