The capacity of stream biofilms to transform and assimilate N in highly N-loaded streams is essential to guarantee the water quality of freshwater resources in urbanized areas. However, the degree of N saturation experienced by urban streams and their response to acute increases in N concentration are largely unknown. We measured changes in the rates of NH4+ uptake (UNH4) and oxidation (UAO) resulting from experimental increases in NH4+-N concentration in mature biofilms growing downstream of a wastewater treatment plant (WWTP) and, thus, naturally exposed to high N concentration. We investigated the responses of UNH4 and UAO to NH4+-N increases and the abundance of NH4+ oxidizing bacteria and archaea (AOB and AOA) in epilithic and epipsammic biofilms. UNH4 and UAO increased with increasing NH4+-N concentration for the 2 biofilm types, suggesting no N saturation under ambient levels of NH4+-N. Thus, these biofilms can contribute to mitigating N excesses and the variability of NH4+-N concentrations from WWTP effluent inputs. The 2 biofilm types exhibited different Michaelis-Menten kinetics, indicating different capacity to respond to acute increases in NH4+-N concentration. Mean UNH4 and UAO were 5× higher in epilithic than epipsammic biofilms, coinciding with a higher abundance of AOA+AOB in the former than in the later (76 × 104 vs 14 × 104 copies/cm2). AOB derived from active sludge dominated in epilithic biofilms, so our results suggest that WWTP effluents can strongly influence in-stream NH4+ processing rates by increasing N inputs and by supplying AOA+AOB that are able to colonize some stream habitats