Microbial biomass production has been measured to investigate the contribution of planktonic bacteria to fluxations in dissolved organic matter in marine and freshwater environments, but little is known about biomass production of thermophiles inhabiting geothermal and hydrothermal regions. The biomass production of thermophiles inhabiting an 85°C geothermal pool was measured by in situ cultivation using diffusion chambers. The thermophiles' growth rates ranged from 0.43 to 0.82 day−1, similar to those of planktonic bacteria in marine and freshwater habitats. Biomass production was estimated based on cellular carbon content measured directly from the thermophiles inhabiting the geothermal pool, which ranged from 5.0 to 6.1 μg C l−1 h−1. This production was 2–75 times higher than that of planktonic bacteria in other habitats, because the cellular carbon content of the thermophiles was much higher. Quantitative PCR and phylogenetic analysis targeting 16S rRNA genes revealed that thermophilic H2-oxidizing bacteria closely related to Calderobacterium and Geothermobacterium were dominant in the geothermal pool. Chemical analysis showed the presence of H2 in gases bubbling from the bottom of the geothermal pool. These results strongly suggested that H2 plays an important role as a primary energy source of thermophiles in the geothermal pool.