Spider-Man leaves his "friendly" neighborhood of New York City to embark on a side-scrolling adventure set in such exotic locales as Nepal, Tokyo, Egypt, and Transylvania. Throughout his travels, Spidey must physically "convince" some of his greatest enemies to help him defeat a more pervasive threat to the world: a vast army of symbiotes. The developers borrowed elements from the film trilogy as well as the comic book series to create a vibrant, cartoon-like art style designed to appeal to younger audiences. One of Friend or Foe's key features is its cooperative play mode, in which a nearby friend can enter the fray via local wireless connection and start pummeling enemies.

The DS version has Spider-Man eventually calling on the help of seven potential sidekicks. Playable enemies include Green Goblin, Doc Ock, and Venom, while friendly companions include Blade and Black Cat. The action sequences involve using each character's distinct fighting moves and special attacks to eliminate groups of onscreen foes. Solo players can instantly switch between Spidey and his selected sidekick, allowing for specific team-up and combo moves. Hidden throughout each 3D world are a number of Spider Tokens to unlock bonus content or to purchase upgrades. The action is displayed on both DS screens, allowing players a cross-section view of the area to help find hidden tokens or to more easily track boss characters. The touch screen is also used in mini-game-style events such as picking locks, activating computer terminals, defusing bombs, and more.