The plot of Black Mesa is almost identical to Half-Life's storyline, playable through the "Lambda Core" chapter. As in the original game, the player controls Gordon Freeman, a scientist working at the Black Mesa Research Facility. He is tasked to place a sample of a strange material into an electromagnetic instrument, using the Hazardous Environment Suit Mark IV to do so safely. However, the sample material causes a "resonance cascade", devastating the facility and creating an interdimensional rift to an alien dimension called Xen, bringing its alien creatures to Earth.

Freeman survives, finds other survivors, and makes his way to the surface with the protection of his hazard suit to get help. Upon reaching the surface, however, he finds that the facility is being cleansed of any living thing - human or alien - by armed forces. From other scientists, Freeman finds the only way to stop the alien invasion is to cross over to Xen and destroy the entity holding the portal open.