The hyporheic zone, where surface water and groundwater mix, is an important microbial habitat where biogeochemical reactions influence water quality. We show that spatial variability in hyporheic flow in the East River near Crested Butte, Colorado, drives heterogeneity in streambed geochemical conditions and microbial community assemblages, but the diversity of assemblages remains nearly constant throughout the reach. In July 2018, we collected approximately 100 pore water samples at 20 cm depth and analyzed them for anions, cations, dissolved organic carbon, dissolved organic matter (DOM) quality, and basic water quality parameters. Vertical hydraulic head gradients were also measured to assess the potential for upward or downward flow, and heat tracing was used to quantify vertical flux rates at a subset of locations. We found that regions of the streambed that are more groundwater-dominated contain less dissolved oxygen, higher concentrations of reduced metals, and more microbially-processed, recalcitrant DOM, while more surface water-dominated locations contain higher dissolved oxygen concentrations and terrestrially-derived, labile DOM. 16S rRNA gene sequencing of extracted DNA revealed that microbial community composition varies with geochemical gradients related to hyporheic flow. These findings provide a better understanding of hyporheic controls on streambed biogeochemistry during the baseflow season, which is expected to lengthen with climate change in alpine watersheds due to earlier snowmelt onset and reduced snowpack.