Since the first discovery of macrofaunal and microbial communities endemic to hydrothermal vents, chemolithoautotrophic microorganisms at and beneath the seafloor have attracted the interest of many researchers. This type of microorganism is known to obtain energy from inorganic substances (e.g., reduced sulfur compounds, molecular hydrogen, and methane) derived from subsurface physical and chemical processes, such as water-rock interactions. As the primary producers, they sustain chemosynthetic ecosystems, which are fundamentally different from terrestrial and shallow marine ecosystems that are sustained by photosynthetic primary production. It is possible that the chemosynthetic ecosystems at and beneath the seafloor are vast and metabolically active, playing an important role in the global geochemical cycles of many bio-essential elements. However, even today, the spatial and temporal distributions of the unseen chemosynthetic biosphere are largely uncertain. Here, we present geochemical constraints on the estimate of the potential biomass in seafloor and subseafloor chemosynthetic ecosystems sustained by high-temperature deep-sea hydrothermal activities and low-temperature alteration/weathering of oceanic crust. The calculations are based on the fluxes of metabolic energy sources (S, H 2 , Fe, and CH 4 ), the chemical energy yields for metabolic reactions, and the maintenance energy requirements. The results show that for deep-sea hydrothermal vent ecosystems, most bioavailable energy yields (86 %) are due to oxidation reactions of S. In contrast, for subseafloor oceanic crust ecosystems, oxidation reactions of Fe and S generally yield the same amounts of bioavailable energy (59 % and 41 %, respectively). The estimated biomass potential in the subseafloor oceanic crust ecosystems (0.14 Pg C) is one order of magnitude higher than that in global deep-sea hydrothermal vent ecosystems (0.0074 Pg C), most likely reflecting the greater flux of