Serious Sam II will feature NPCs in every one of its seven worlds, across which there are 40 different levels. Some of the types of worlds to be seen in the final game include environments of jungles, lava, a futuristic city, and an oriental city. Croteam and 2K Games expect Serious Sam II to take the average gamer 25-30 hours to complete, and it will include a full two hours of cutscenes, featuring the wacky story to be expected from Croteam. NPCs can be allies to Sam, and in one of the worlds they provide Sam with the parrot weapon. NPCs will be able to man turrets, fight against Sam’s enemies, and even ride in vehicles alongside Sam. Some of the vehicles in Serious Sam II include speeder bikes, surfboards, two-person vehicles, animals which can take out enemies for you, enemies which can be coerced into being ridden, and a spiked ball similar to a hamster ball that can be used to crush over enemies. Also shown was a hover speeder which had some really cool looking jet trails on it, and had plasma rifles as a weapon as well as being able to run down enemies.

The enemies of Serious Sam II include some of the original baddies such as suicide bombers, harpies, and kleers, all of which have been upgraded for the sequel. New enemies include a large wind-up rhino, similar to the original werebull, metallic spiders, a giant raptor on mechanized legs, and football orc-looking creatures that charge Sam with spikes and throw flaming explosive footballs. Dropships are a new addition to Serious Sam II, and will be used at times as a replacement to simply spawning enemies. This allows the player the ability to shoot down a dropship before it can unload all the enemies, which can make things simpler for the player. Netricsa, the GUI , is now a woman in cutscenes, and Croteam presents interactions between Sam and Netricsa as a parody of soap operas.