On the Trinity Church's clock, the hands are set at 1:21. This is a reference to 2014's The Amazing Spider-Man 2 movie, when the clock tower's hands stopped spinning rapidly and position on 1:21, after Gwen Stacey died from a fall. This is also a reference to the story "The Night Gwen Stacy Died" appearing in the comic The Amazing Spider-Man issue #121. Additionally, the colored green and purple windows matches the Green Goblin's costume.

Found on one of the movie theater marquees is the message "Maddie, Will You Marry Me?" This was an Easter egg included in the game at the request of fan Tyler Schultz who tweeted out to Insomniac asking them for help with his marriage proposal to his then girlfriend of 5 years Madison Gamble ("Maddie" for short) by including the message somewhere in-game. However, by the time the game had released, Gamble had broken up with him with Schultz explaining what had happened. Insomniac had offered to patch out the message but Schultz requested to keep it in as a reminder for "the almost mistake that I made." adding that his girlfriend told him "that the video game message wasn’t the way she wanted to be proposed to, either."

Gamble would later say in a statement that she never even liked video games and only tolerated them because of Schultz. She claimed that she broke up with Schultz due to his behavior and gave him a month to change but to no avail.

When the player uses the Holo Decoy gadget, holographic clones of Spider-Man will appear to distract enemies and say various phrases. In the Japanese version of the game, one phrase he will say is "The Emissary from Hell - Spider-Man". This is a reference to Spider-Man's catchphrase from the 1978 Spider-Man Japanese television series produced by Toei.

According to Steven L Kent’s book The Ultimate History of Video Games Vol 2, executive vice president and head of Marvel Games Jay Ong stated that after they terminated their licensing deal with Activision he offered the exclusivity deal to Microsoft, but it was ultimately turned down. Microsoft‘s strategy at the time was to focus on its own IP, and as such, it decided to pass on the offer.

In an October 24, 2023 WIRED video/interview with the Marvel's Spider-Man series creative director Bryan Intihar, he revealed that in order to make the sound effects for the web shooters, the audio team used silly string, plastic wrap, shrink wrap and even made the "thlip" sound with their voices, all of which were integrated into making their own webbing sound effects.

In a 2018 Syfy Wire interview with writers Christos Gage and Jon Paquette, they revealed that Silver Sable and Sable International were included in the game because they felt that there needed to be a presence or force in the overworld that added more stakes and tension to the game once the story events "became dire".

The Spider-Man and Wolverine games developed by Insomniac Games are established to take place in Earth-1048 in Marvel's multiverse. This reality designation was first established in the 2018 comic miniseries "Spider-Geddon", which canonically takes place sometime after the events of the first Spider-Man game.