Stream biofilms have the capacity to modify the passage of macronutrients through catchments as they respond to nutrient compositions and ratios from different sources. Knowledge of coupled cycling of N, P and organic C in flowing freshwaters is essential to understanding and predicting aquatic ecosystems responses to environmental change comprising multiple chemical and physical stressors. Colonisation on nutrient diffusing substrates (glucose-C, inorganic NP, combined CNP and control applied in-situ in an oligotrophic, upland stream) led to biofilms differing in community and element compositions. The 72 biofilms were transferred to replicated recirculating water chambers (1 L volume) for 4-days where additional effects of light and temperature treatments were investigated on nutrient exchange with the water column. Chemical (nutrient analyses, ¹³C, ¹⁵N tracing, stoichiometry) and biological (chlorophyll, TRFLP) analyses were performed to understand the biofilm composition changes and interaction with the water column. Biofilms combining C with NP incorporated more N and P relative to controls than did those with NP alone. During the chamber phase C-treated biofilms resulted in lower water column N, P concentrations with CNP relative to NP treatments. The effects of the light and temperature were manifested mainly in impaired net nutrient uptake at temperature deviating from ambient stream temperatures. The effects of organic C on N, P cycling (and vice-versa) in mixed biofilms and their interaction with waters is a developing field. Combining in-stream and chamber tests has shown potential for studying in controlled and replicated systems such complex interactions.