Phototrophic river biofilms are microbial assemblages involved in in-stream processes. For a better understanding of N-cycling, the denitrifier community abundance, activity and structure were monitored in natural biofilm assemblages, in two sites exhibiting contrasting nutrient concentrations during a 1-year period. The denitrifier abundance, estimated by quantification of the nirS, nirK and nosZ genes, correlated to biofilm biomass and total bacterial counts. Site-related differences in denitrification activity were observed and the rates were significantly correlated with the nosZ gene copy numbers and biofilm biomass. The denitrifier community structure, assessed by PCR-DGGE of nosZ, differed between sites with only minor differences between sampling occasions, and correlated with the total bacterial community structure. Altogether, these findings suggest that nutrient loading, especially nitrogen, affect both denitrifier community structure and activity.