ABSTRACT The marine subsurface is a long-term sink of atmospheric carbon dioxide with significant implications for climate on geologic timescales. Subsurface microbial cells can either enhance or reduce carbon sequestration in the subsurface, depending on their metabolic lifestyle. However, the activity of subsurface microbes is rarely measured. Here, we used nanoscale secondary ion mass spectrometry (nanoSIMS) to quantify anabolic activity in 3,203 individual cells from the thermally altered deep subsurface in the Guaymas Basin, Mexico (3–75 m below the seafloor, 0–14°C). We observed that a large majority of cells were active (83%–100%), although the rates of biomass generation were low, suggesting cellular maintenance rather than doubling. Mean single-cell activity decreased with increasing sediment depth and temperature and was most strongly correlated with porewater sulfate concentrations. Intracommunity heterogeneity in microbial activity decreased with increasing sediment depth and age. Using a dual-isotope labeling approach, we determined that all active cells analyzed were heterotrophic, deriving the majority of their cellular carbon from organic sources. However, we also detected inorganic carbon assimilation in these heterotrophic cells, likely via processes such as anaplerosis, and determined that inorganic carbon contributes at least 5% of the total biomass carbon in heterotrophs in this community. Our results demonstrate that the deep marine biosphere at Guaymas Basin is largely active and contributes to subsurface carbon cycling primarily by not only assimilating organic carbon but also fixing inorganic carbon. Heterotrophic assimilation of inorganic carbon may be a small yet significant and widespread underappreciated source of labile carbon in the global subsurface. IMPORTANCE The global subsurface is the largest reservoir of microbial life on the planet yet remains poorly characterized. The activity of life in this realm has implications for long-term elemental cycling, particularly of carbon, as well as how life survives in extreme environments. Here, we recovered cells from the deep subsurface of the Guaymas Basin and investigated the level and distribution of microbial activity, the physicochemical drivers of activity, and the relative significance of organic versus inorganic carbon to subsurface biomass. Using a sensitive single-cell assay, we found that the majority of cells are active, that activity is likely driven by the availability of energy, and that although heterotrophy is the dominant metabolism, both organic and inorganic carbon are used to generate biomass. Using a new approach, we quantified inorganic carbon assimilation by heterotrophs and highlighted the importance of this often-overlooked mode of carbon assimilation in the subsurface and beyond.