The Second Encounter starts right where the previous chapter left off, with Sam travelling to Sirius on the starship. Unfortunately Sam's vehicle is accidentally hit by the "Croteam crate-bus" and plummets down to Earth's surface. Serious Sam crash lands in Central America in the Mayan age. However not all is lost, since the Sirians left a back-up ship on Earth. Since this vehicle is located in a different age from the one Sam finds himself, the hero has to uncover the locations of a number of Sirian time-gates that will help him reach his destination. With this objective Sam storms through Mesoamerica, then travels to Mesopotamia and finally to medieval Europe where the starship is located. Finally, in front of the Cathedral of Holy Blood, Sam confronts Mordekai The Summoner, the last obstacle to overcome on his path to the Holy Grail. After a time-consuming battle with Mordekai's minions, The Summoner is finally silenced forever. In the cathedral, Sam lifts the Holy Grail, followed by his confession of his sins in a booth... to Mental, during which he reassures Mental that "he's coming to get him."

Other than the more varied locations, The Second Encounter features some additional weapons — a chainsaw, a flamethrower, a sniper rifle, and the powerful Serious Bomb — and a number of new monsters that Sam has to face. This game has also seen the release of an expansion titled Serious Sam: Dark Island, featuring some new levels for single and multiplayer mode.

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