Seasonal changes in the abundance of bacteria belonging to different phylogenetic groups in epilithic biofilms from a northeastern Ohio (USA) stream were examined using fluorescent in situ hybridization (FISH). Changes over a 13-mo period were observed in the biofilm assemblages, dominated by α- and β-proteobacteria. Numbers of β-proteobacteria and Cytophaga–Flavobacterium peaked during the winter months and coincided with increased NO₃ concentration. Actinobacteria (Gram-Positive bacteria with high guanine and cytosine [GC] content) had no relationship with any measured environmental variable and accounted for <3% of the overall bacterial assemblage. Abundance of 3 bacterial species examined, Acinetobacter calcoaceticus, Burkholderia cepacia, and Pseudomonas putida, was similar, except in the summer when numbers of B. cepacia were higher than the other 2 species. Detrended correspondence analysis extracted 2 factors that explained 69.2% of the total variation. β-proteobacteria and Cytophaga–Flavobacterium clustered with conductivity and concentrations of NO₃, dissolved organic C, and soluble reactive P in the 1st factor, while A. calcoaceticus, B. cepacia, and P. putida clustered with temperature and turbidity in the 2nd factor. Our study revealed large seasonal fluctuations in the abundance of the different bacterial taxa examined in biofilms, and also demonstrated the potential influences of various environmental variables on microbial community composition in aquatic systems.