We report on the effect of the end-Cretaceous impact event on the present-day deep microbial biosphere at the impact site. IODP-ICDP Expedition 364 drilled into the peak ring of the Chicxulub crater, México, allowing us to investigate the microbial communities within this structure. Increased cell biomass was found in the impact suevite, which was deposited within the first few hours of the Cenozoic, demonstrating that the impact produced a new lithological horizon that caused a long-term improvement in deep subsurface colonization potential.