Inorganic nitrogen (N) fertilizer, generally applied as anhydrous ammonia, enhances crop yields, but runoff from agricultural fields to streams can degrade water quality. Ammonium can be removed from the water column via assimilatory uptake by stream biofilms or can be transformed to nitrate via the dissimilatory process of nitrification. We examined the effects of ammonium and carbon (C) availability on ammonium uptake, nitrification, and reach-scale metabolism using replicated nutrient additions to four experimental streams at the Notre Dame Linked Experimental Ecosystem Facility (ND-LEEF). We compared uptake and transformation rates across a biofilm colonization sequence (i.e., early and late biofilm growth, then senescence) and under varying light conditions (i.e., day vs. night). We conducted short-term releases of ammonium and ammonium+C (added as acetate), and quantified reach-scale nitrification rates by documenting simultaneous ammonium removal and nitrate production along each stream. We also estimated reach-scale gross primary production (GPP), ecosystem respiration (ER), and gas exchange (k) using miniDOT sensors. We measured significant declines in ammonium during all releases (n=48), and ammonium uptake varied across biofilm colonization phases and light conditions. However, reach-scale nitrification was only detectable in 18% of releases, thus assimilatory uptake was the dominant mechanism for ammonium removal. When detectable, nitrification rates were higher during the late biofilm phase (1.6±0.3 mg N m-2 h-1) compared to early biofilm (0.05 mg N m-2 h-1). There was no measurable nitrification during senescence, and both GPP and ER were significantly lower during this phase (Kruskal-Wallis p<0.001 and p<0.05, respectively). Our results suggest that biofilm constituents (both autotrophs and heterotrophs) consistently assimilate ammonium, but outcompete nitrifying bacteria, thereby limiting nitrification, even when ammonium availability is high. This research fills critical gaps regarding controls on stream nitrification rates and helps improve understanding about how human-impacted headwater streams respond to elevated ammonium.