Mesoscale spatial (centimeters–meters) and temporal (days–weeks) flow variability plays an important role in stream ecosystem structure and function. The structural effects of flow variability have been widely tested but its influence on ecosystem processes is less well understood. Our study examined the influence of mesoscale flow variability on cobble biofilm metabolism in an Australian regulated upland stream that had been subjected to severe catchment disturbance as a result of extensive fire 10 mo before the study. Rates of primary production and respiration were calculated from changes in dissolved O₂ over 24 h in benthic chambers containing stream cobbles sampled before and after high-discharge events in areas with differing surface-flow types. Overall, cobble biofilm communities were strongly autotrophic (primary production » respiration), probably because of reduced shading and increased nutrient influx caused by the recent fire. Differences in production and respiration before and after individual high-discharge events were inconsistent and, therefore, not statistically significant. The effect of high discharge on respiration was greater when high-discharge events were preceded by long periods of low discharge, but the number of events sampled was limited and this result could not be tested statistically. Rates of respiration and concentrations of organic material, chlorophyll a, and pheophytin a also varied spatially, and these variables were affected more by mesoscale variation in hydraulic conditions than by reach-scale variation in hydraulic conditions. Our results suggest that flow management has the potential to alter benthic metabolism.