The first step of nitrification, the oxidation of ammonia to nitrite, is important for reducing eutrophication in freshwater environments when coupled with anammox (anaerobic ammonium oxidation) or denitrification. We analyzed active formerly biofilm-associated aerobic ammonia-oxidizing communities originating from Ammerbach (AS) and Leutra South (LS) stream water (683 ± 550 [mean ± standard deviation] and 16 ± 7 µM NH4+, respectively) that were developed in a flow-channel experiment and incubated under three temperature regimens. By stable-isotope probing using 13CO2, we found that members of the Bacteria and not Archaea were the functionally dominant autotrophic ammonia oxidizers at all temperatures under relatively high ammonium loads. The copy numbers of bacterial amoA genes in 13C-labeled DNA were lower at 30°C than at 13°C in both stream enrichment cultures. However, the community composition of the ammonia-oxidizing bacteria (AOB) in the 13C-labeled DNA responded differently to temperature manipulation at two ammonium concentrations. In LS enrichments incubated at the in situ temperature (13°C), Nitrosomonas oligotropha-like sequences were retrieved with sequences from Nitrosospira AmoA cluster 4, while the proportion of Nitrosospira sequences increased at higher temperatures. In AS enrichments incubated at 13°C and 20°C, AmoA cluster 4 sequences were dominant; Nitrosomonas nitrosa-like sequences dominated at 30°C. Biofilm-associated AOB communities were affected differentially by temperature at two relatively high ammonium concentrations, implicating them in a potential role in governing contaminated freshwater AOB distributions.