Uptake and storage by biofilms facilitate the ability of streams to act as sinks for inorganic nutrients. Quantifying P fluxes over short time periods is important for understanding physiological responses to nutrient inputs, but few researchers have examined spatial and temporal variability in patterns of short-term biofilm nutrient uptake. We sampled intact stream biofilm assemblages seasonally (autumn—summer) across a spatial productivity gradient (8 streams in 2 provinces [Plateau and Piedmont] in Pennsylvania, USA) and tested responses to a P source over the first few minutes of exposure to resolve changes in initial assimilatory kinetics in short-term radiotracer experiments. We estimated P uptake and efflux rates with local regression (LOESS) and found distinct breakpoints in P fluxes. Mean maximum uptake occurred at 1.65 and 2.60 min, whereas maximum efflux occurred at 4.51 and 4.62 min in Piedmont and Plateau provinces, respectively. Biofilm nutrient content and accumulation rates were strong predictors of P uptake and efflux. P uptake and efflux differed between provinces (p = 0.005) and among seasons (p = 0.001). Our results show that considerable and rapid exchange processes occur at early time periods (<5 min). The magnitude of these exchange processes seems to diminish over longer periods (15–30 min), indicating that nutrient processing is a nearly instantaneous physiological, dynamic process. Thus, experimental time periods that are hours long or longer may obscure essential short-term responses. Spatio-temporal effects (productivity and seasonality) are strong determinants of P fluxes.