N₂-fixing bacteria convert atmospheric N₂ gas to biologically available forms, yet N limits primary production in many ecosystems. Despite an abundant source of N, other elements may limit N input via N₂-fixation in benthic environments. We examined how N, P, and the micronutrients Fe and Mo (cofactors of nitrogenase) might affect N₂-fixation. We used short-term nutrient additions to assess macronutrient effects on N₂-fixation of intact epilithic biofilm in Ditch Creek, Wyoming (USA). Additions of NH₄ , NO₃⁻, and PO₄³⁻ at varying concentrations did not alter N₂-fixation over 4 h. We assessed macro- and micronutrient effects on N₂-fixation, biofilm biomass, and composition of the biofilm algal assemblage at Ditch, Spread, and Kimball Creeks, Wyoming, via 6-wk nutrient amendments with nutrient-diffusing substrata. Fe and Mo additions did not affect N₂-fixation or biofilm biomass, but NO₃⁻ and PO₄³⁻ altered the biofilm biomass, algal assemblage structure, and N₂-fixation. N and P colimited biofilm biomass in Ditch Creek, and P limited biofilm biomass in Spread Creek. N₂-fixation was inhibited by NO₃⁻ and stimulated by PO₄³⁻. Inhibition by NO₃⁻ was stronger than stimulation by PO₄³⁻. Trends in accumulation of N₂-fixers, mostly Epithemia sorex with N₂-fixing endosymbionts, corresponded to effects of nutrient additions on N₂-fixation. The algal assemblage shifted from N₂-fixer dominance in control and P treatments to fewer N₂-fixers and increased biovolume of nonN₂-fixing diatoms in the N and N P treatments. N₂-fixation/unit E. sorex biovolume was inhibited by NO₃⁻ and stimulated slightly by PO₄³⁻. A combination of demographic and mutualistic responses to nutrients altered N input via N₂-fixation.