Hydrothermal fluids passing through basaltic rocks along mid-ocean ridges are known to be enriched in sulfide, while those circulating through ultramafic mantle rocks are typically elevated in hydrogen. Therefore, it has been estimated that the maximum energy in basalt-hosted systems is available through sulfide oxidation and in ultramafic-hosted systems through hydrogen oxidation. Furthermore, thermodynamic models suggest that the greatest biomass potential arises from sulfide oxidation in basalt-hosted and from hydrogen oxidation in ultramafic-hosted systems. We tested these predictions by measuring biological sulfide and hydrogen removal and subsequent autotrophic CO2 fixation in chemically distinct hydrothermal fluids from basalt-hosted and ultramafic-hosted vents. We found a large potential of microbial hydrogen oxidation in naturally hydrogen-rich (ultramafic-hosted) but also in naturally hydrogen-poor (basalt-hosted) hydrothermal fluids. Moreover, hydrogen oxidation–based primary production proved to be highly attractive under our incubation conditions regardless whether hydrothermal fluids from ultramafic-hosted or basalt-hosted sites were used. Site-specific hydrogen and sulfide availability alone did not appear to determine whether hydrogen or sulfide oxidation provides the energy for primary production by the free-living microbes in the tested hydrothermal fluids. This suggests that more complex features (e.g., a combination of oxygen, temperature, biological interactions) may play a role for determining which energy source is preferably used in chemically distinct hydrothermal vent biotopes.