Microbes mediate important biogeochemical processes such as nutrient cycling and carbon mineralization in lotic ecosystems. Time and cost constraints, coupled with the assumption that microbial communities are functionally redundant have historically led to microbes being left in the black box in ecosystem models. However, advances in sequencing as well as studies demonstrating the functional significance of distinct microbial communities have shifted the paradigm. Still, the factors controlling the composition of microbial communities remain unresolved. This study investigated how land use, water quality, and sediment chemistry influence benthic microbial community composition in temperate streams of the northeastern USA. We hypothesized that across a mixed land-use watershed, distinct microbial communities would exist within headwater streams draining agricultural, forested, and residential watersheds. We predicted these distinct microbial communities would be associated with differences in water quality and sediment chemistry variables such as nutrient concentrations, pH, sediment C:N ratios, and suspended sediment load. To test our hypothesis, we collected benthic sediment samples and water quality samples in headwater streams representing a wide gradient in land use, water, and sediment chemistry. Bacterial and fungal community composition was analyzed using marker gene DNA sequencing. We found bacterial community composition to be strongly influenced by land use, with communities in forested streams differing substantially from those in agricultural and residential streams. Further, we found community diversity declines with percent of agriculture and residential land within a watershed. This study demonstrates how watershed scale factors exert influence over benthic microbial communities. Future work should examine how function differs in these distinct microbial communities.