The wellbeing of freshwater ecosystems relies on critical biogeochemical processes facilitated by aquatic microbial communities. In a stream, many of these processes may occur in the hyporheic zone, an ecotone located beneath the stream bed where surface water and groundwater mix. Microbial communities in the hyporheic zone attenuate contaminants, facilitate nutrient turnover in a stream, and drive up to 95% of whole-river metabolism. Despite this, our understanding of hyporheic microbial communities is generally limited, and their significance is underemphasized in river research and conservation. In this study, we sampled water from the hyporheic zone for its geochemical properties and microbial diversity at sites along the Llano and Concho Rivers, tributaries of the Colorado River in central Texas (USA) that had contrasting flow regimes (high vs. low flow, respectively) and sediment properties (cobble vs. sand/silt, respectively). Microbial diversity was characterized from 16S rRNA gene sequence analysis and their ecologically relevant traits using FAPROTAX, a tool used to predict metabolic functions. Results from the rivers were compared to assess the effects of geochemistry, flow, and sediment properties on community diversity and functionality to advance our understanding of hyporheic microbial processes, and improve our ability to predict how future ecological changes may alter microbial communities and how that, ultimately, will impact river functionality.