Headwater streams are foci for nutrient and energy loading from terrestrial landscapes, in situ nutrient transformations, and downstream transport. Despite the prominent role that headwater streams can have in regulating downstream water quality, the relative importance of processes that can influence nutrient uptake have not been fully compared in heterotrophic aquatic systems. To address this research need, we assessed the seasonality of dissolved organic carbon (DOC) and nitrate (NO₃⁻) uptake, compared the relative influence of hydrologic and biogeochemical drivers on observed seasonal trends in nutrient uptake, and estimated the influence of these biological transformations on watershed scale nutrient retention and export. We determined that seasonal reductions in DOC and NO₃⁻ concentrations led to decreases in the potential for the biotic community to take up nutrients, and that seasonality of DOC and NO₃⁻ uptake was consistent with the seasonal dynamics of ecosystem metabolism. We calculated that that during the post-snowmelt period (June to August), biotic retention of both dissolved organic carbon and nitrate exceeded export fluxes from this headwater catchment, highlighting the potential for biological processes to regulate downstream water quality.