Watershed urbanization introduces a variety of physical, chemical, and thermal stressors to receiving streams and leads to well-documented declines in the diversity of fish and macroinvertebrates. Far less knowledge is available about how these urban stressors affect microbial communities and microbially mediated ecosystem properties. We examined 67 chemical, physical, and biological attributes of streams draining 47 watersheds in the metropolitan area surrounding Raleigh, North Carolina. Watersheds ranged from undeveloped to 99.7% developed watershed area. In contrast to prior investigators, we found no consistent changes in habitat structure, channel dimensions, or bed sediment size distributions along the urbanization gradient. Watershed urbanization led to large and consistent changes in receiving stream chemistry (increases in NO 3 2 , bioavailable and algal-derived dissolved organic C, and the trace metals Pb, Cd, and Zn) and thermal regimes. These chemical and thermal changes were not associated with any consistent shifts in microbial community structure or taxonomic richness, based on terminalrestriction fragment length polymorphism and pyrosequencing methods, despite the fact that these urban stressors were associated with commonly reported declines in macroinvertebrate taxonomic richness and altered macroinvertebrate community composition. Chemical and thermal changes as a function of % developed watershed area also were unrelated to shifts in microbially mediated biogeochemical processes (C mineralization, denitrification potential, and substrate-induced respiration). A broad urbanization gradient sampled in this region suggests that stream ecosystem responses to watershed urbanization can follow diverse trajectories.