The composition, growth and metabolic processes of the autotrophic portion of stream biofilm communities is governed by resource availability. Bioavailable nitrogen (NO3-, NH4-) and phosphorus (PO43-) fuel primary production, while micronutrients play a lesser-understood role in the enzymes that facilitate specific physiological processes such as organic nutrient uptake and assimilation. Without micronutrients, cycling and assimilation of organic macronutrients, photosynthesis and respiration within biofilms would not be possible. Nutrient availability also shapes algal community composition by selecting for particular taxa. Using nutrient diffusing substrate, we assessed the role of macro (N, P) and micronutrients (Zn, Mo) on stream biofilm primary producer diversity, growth, metabolism and alkaline phosphatase activity (APA) across four sites upstream and downstream of a wastewater treatment plant (WWTP) and nutrient rich tributary. Results indicate that APA, which facilitates organic P acquisition, increased for N treatments across all sites, while algal diversity had greater inter-site variation than variation elicited by nutrient treatment. Phosphorus enrichment increased growth prior to input from the WWTP, while growth had a negative response to N treatment directly after the WWTP inflow. Upstream of a nutrient rich tributary, N+P and N+Zn treatments stimulated the most growth, suggesting co-limitation of N and P and alleviation of P limitation by Zn through increased APA. These results indicate that macro and micronutrients co-limit specific processes within biofilms, and that micronutrients may regulate organic nutrient cycling in stream biofilms.