Biofilms, complex microbial communities within extracellular matrices, play a critical role in regulating biogeochemical processes in streams, including cycling of organic matter and nutrients. While much of the research on biofilm nutrient limitation focuses on C, N, and P, organisms require a wider range of elements, including trace metals, which are essential cofactors in enzymes that drive biogeochemical cycles. Some extracellular enzymes secreted by biofilms for the degradation of organic matter (OM) require metal cofactors. This research expands the ecoenzymatic stoichiometry framework, traditionally centered on C, N, and P, by incorporating trace metals. Biofilm samples from 20 streams along an urban-to-forested gradient were analyzed for the activities of β-glucosidase (BG), leucine aminopeptidase (LAP), β-1,4-N-acetylglucosaminidase (NAG), and alkaline phosphatase (AP). Stream metal concentrations were measured from filtered water samples and diffusive gradient thin films (DGT). Geospatial analyses were applied linking land use, water chemistry, and ecoenzymatic rates. Rates of BG showed a stronger relationship with AP (log-log slope=0.67) than NAG (log-log slope= 0.54), which suggests that P is more commonly limiting in our study streams than N. Urban streams exhibited less AP activity (167 μmol/hr) compared to mixed (253 μmol/hr) and forested streams (396 μmol/hr), which suggests that P limitation is alleviated by human inputs.Additionally, BG showed a weak negative correlation with DGT Co concentrations (R²=0.23, p=0.03), suggesting interactions with carbonic anhydrase (CA), a Co-bearing metalloenzyme. CA catalyzes the inter-conversion of HCO3− and CO2, bypassing the need for heterotrophs to acquire C from OM using BG. Negative correlations between AP and Cd, Mo, B, V, and As were also observed, though mechanisms remain unclear. These findings underscore trace metals' influence on biofilm enzymatic activities, revealing a need for further research on their role in regulating biogeochemical processes.