<p>Patch-scale heterogeneity in stream biota may be explained by upwelling groundwater interacting with stream water to generate distinct microhabitats at the streambed. We examined the effects of variable groundwater upwelling on among-patch heterogeneity in stream biota and processes in a nutrient-rich stream. Fifty sampling points exhibiting variable groundwater upwelling rates were established in a headwater stream reach in Ontario, Canada, based on past surveys of streambed temperatures. At each sampling point, biofilm and algal growth, diatom assemblages, and cellulose decomposition were measured using artificial substrates on and beneath the streambed. Groundwater upwelling variation had only modest influence on near-bed stream water conditions (i.e. temperature and water chemistry), likely due to rapid dilution by stream water. Stream velocity, and not groundwater upwelling, was the primary determinant of spatial patterns of stream biofilm structure and function on the streambed surface. Subsurface temperatures, and thus, groundwater upwelling patterns, were strongly associated with subsurface cellulose decomposition. Groundwater upwelling rates and/or differences in water quality (e.g. nutrient concentrations) between groundwater and stream water may therefore need to be substantial to significantly influence near-bed conditions and associated patch scale patterns of stream biofilms in nutrient-rich agricultural streams.</p>