Below the seafloor at deep-sea hot springs, mixing of geothermal fluids with seawater supports a potentially vast microbial ecosystem. Although the identity of subseafloor microorganisms is largely known, their effect on deep-ocean biogeochemical cycles cannot be predicted without quantitative measurements of their metabolic rates and growth efficiency. Here, we report on incubations of subseafloor fluids under in situ conditions that quantitatively constrain subseafloor primary productivity, biomass standing stock, and turnover time. Single-cell-based activity measurements and 16S rRNA-gene analysis showed that Campylobacteria dominated carbon fixation and that oxygen concentration and temperature drove niche partitioning of closely related phylotypes. Our data reveal a very active subseafloor biosphere that fixes carbon at a rate of up to 321 μg C⋅L−1⋅d−1, turns over rapidly within tens of hours, rivals the productivity of chemosynthetic symbioses above the seafloor, and significantly influences deep-ocean biogeochemical cycling.