Studies on the role of limiting elements on primary producers in streams have been largely focused on nitrogen (N) and phosphorus (P). However, trace nutrients can mediate ecological processes like photosynthesis, respiration, and nutrient cycling but limitation by trace metals is not fully understood. Assessing the stoichiometry of stream organisms, along with water chemistry, under macro- and micronutrient limiting conditions could facilitate more understanding of the role of trace metals in streams. We collected biofilm samples from natural inorganic substrates (cobbles, gravels) and wood from 13 streams in Michigan, Ohio, and Virginia, where nutrient-diffusing substrata (NDS) were deployed to test for metal and macronutrient co-limitation. Biofilm samples were digested and analyzed for C, N, P, trace metals (Zn, Fe, Mo, Co, Ni, Mn), and elemental ratios were calculated. Individual biofilm stoichiometry C, N, and P were relatively balanced (i.e., close to the Redfield ratio) in 9 streams. Streams with low iron quota biofilms (Fe:N) were associated with strong response to NDS with combined treatment of N, P, and Fe but not with N or P alone. This result suggests that iron, essential for many microbial metabolisms may be co-limiting with macronutrients in streams with low bioavailable Fe. NDS supplying Zn increased biomass in 46.1% of streams, but there was no relationship between the growth response and biofilm zinc-macronutrient ratios or Zn concentrations in surface water. These results suggest that ambient trace metal availability (especially iron) may modify macronutrient limitation, and lab studies are planned to assess the mechanisms responsible for these relationships.