Mixing processes of reduced hydrothermal fluids with oxygenated seawater and fluid–rock reactions contribute to the chemical signatures of diffuse venting and likely determine the geochemical constraints on microbial life. We examined the influence of fluid chemistry on microbial diversity and activity by sampling diffuse fluids emanating through mussel beds at two contrasting hydrothermal vents. The H2 concentration was very low at the basalt-hosted Clueless site, and mixing models suggest O2 availability throughout much of the habitat. In contrast, effluents from the ultramafic-hosted Quest site were considerably enriched in H2, while O2 is likely limited to the mussel layer. Only two different hydrogenase genes were identified in clone libraries from the H2-poor Clueless fluids, but these fluids exhibited the highest H2 uptake rates in H2-spiked incubations (oxic conditions, at 18 °C). In contrast, a phylogenetically diverse H2-oxidizing potential was associated with distinct thermal conditions in the H2-rich Quest fluids, but under oxic conditions, H2 uptake rates were extremely low. Significant stimulation of CO2 fixation rates by H2 addition was solely illustrated in Quest incubations (P-value <0.02), but only in conjunction with anoxic conditions (at 18 °C). We conclude that the factors contributing toward differences in the diversity and activity of H2 oxidizers at these sites include H2 and O2 availability.