Despite accounting for a significant portion of the Earth’s prokaryotic biomass, controls on the abundance and biodiversity of microorganisms residing in the continental subsurface are poorly understood. To redress this, we compiled cell concentration and microbial diversity data from continental subsurface localities around the globe. Based on considerations of global heat flow, surface temperature, depth and lithology, we estimated that the continental subsurface hosts 2 to 6 × 1029 cells and found that other variables such as total organic carbon and groundwater cellular abundances do not appear to be predictive of cell concentrations in the continental subsurface. Although we were unable to identify a reliable predictor of species richness in the continental subsurface, we found that bacteria are more abundant than archaea and that their community composition was correlated to sample lithology. Using our updated continental subsurface cellular estimate and existing literature, we estimate that the total global prokaryotic biomass is approximately 23 to 31 Pg of carbon C (PgC), roughly 4 to 10 times less than previous estimates.