Epibenthic biofilms are important in regulating nitrogen (N) fluxes in stream ecosystems. The efficiency of the regulation is controlled by hydraulic and biological processes and their interactions. However, knowledge on the underlying physical and biological processes, their controlling parameters, and interactions in stream ecosystems is still limited. To analyze the relative importance of hydraulic and biological controls on biofilm N uptake, we measured turbulence, biofilm N uptake using a stable isotope tracer, and biofilm biomass in two gravel-bed streams with contrasting nutrient concentrations for two seasons. We found high within-stream variability in biofilm areal N uptake and uptake velocity, which exceeded variability between streams and seasons by 60% and 30%, respectively. Sixty-four percent of the within-stream variability in uptake velocity was explained by hydraulic mass transfer and biofilm characteristics, which were described in terms of the turbulent dissipation rate and the biofilm biomass, respectively. We show that surface renewal theory based on scales of the smallest turbulent eddies can be used to estimate transfer velocities at the sediment–water interface and can be extrapolated to larger scales by spatial averaging. Our results improved the mechanistic understanding of the processes regulating biofilm N uptake at small scale which contributes to the understanding of ecosystem functioning in low-order streams and supports upscaling to larger spatiotemporal scales along stream networks.