Nitrate (NO3 -) and ammonium (NH4 +) are the two major dissolved inorganic nitrogen (DIN) species available in streams. Human activities increase stream DIN concentrations and modify the NO3 -:NH4 + ratio. However, few studies have examined biofilm responses to enrichment of both DIN species. We examined biofilm responses to variation in ambient concentrations and enrichments in either NO3 – or NH4 +. We incubated nutrient diffusing substrata (NDS) bioassays with three treatments (DIN-free, +NO3 – and +NH4 +) in five streams. Biomass-specific uptake rates (Uspec) of NO3 – and NH4 + were then measured using in situ additions of 15N-labeled NO3 – and NH4 +. Biomass (estimated from changes in carbon content) and algal accrual rates, as well as Uspec- NO3 – of biofilms in DIN-free treatments varied among the streams in which the NDS had been incubated. Higher ambient DIN concentrations were only correlated with enhanced biofilm growth rates. Uspec-NO3 – was one order of magnitude greater and more variable than Uspec-NH4 +, however similar relative preference index (RPI) suggested that biofilms did not show a clear preference for either DIN species. Biofilm growth and DIN uptake in DIN-amended NDS (i.e., +NO3 – and +NH4 +) were consistently lower than in DIN-free NDS (i.e., control). Lower values in controls with respect to amended NDS were consistently more pronounced for algal accrual rates and Uspec-NO3 – and for the +NH4 + than for the +NO3 – treatments. In particular, enrichment with NH4 + reduced biofilm Uspec- NO3 – uptake, which has important implications for N cycling in high NH4 + streams.