The potential for molecular hydrogen (H-OH- groundwaters bearing up to 4.05 μmol⋅L-1 H2 , 3.81 μmol⋅L-1 methane (CH4) and 946 μmol⋅L-1 sulfate (SO42-) revealed an ecosystem dominated by Bacteria affiliated with the class Thermodesulfovibrionia, a group of chemolithoheterotrophs supported by H2 oxidation coupled to SO42- reduction. In shallower, oxidized Mg2+-HCO3- groundwaters, aerobic and denitrifying heterotrophs were relatively more abundant. High δ13C and δD of CH4 (up to 23.9 ‰ VPDB and 45 ‰ VSMOW , respectively), indicated microbial CH4 oxidation, particularly in Ca2+-OH- waters with evidence of mixing with Mg2+-HCO3- waters. This study demonstrates the power of spatially resolving groundwaters to probe their distinct geochemical conditions and chemosynthetic communities. Such information will help improve predictions of where microbial activity in fractured rock ecosystems might occur, including beyond Earth.