Litterfall is often an important source of organic matter in streams, and biofilms play an essential role in the decomposition and cycling of organic matter in leaves. Biofilms form rapidly on the surfaces of decomposing leaves, but little is known about the taxonomic composition of the bacterial community in leaf-surface biofilms. In addition, it is not known if bacteria in the biofilm colonize leaves from stream water, or if the biofilm forms from growth of bacteria already on the leaf surface. The purposes of our study were to examine the contribution of major taxa of freshwater bacteria to maple-leaf biofilms and to determine if these taxa were present in the biofilm because of colonization from the stream water. Exposed and dialysis-bag-enclosed maple leaves were incubated in a stream in northeastern Ohio during autumn 1998 and 1999. Leaves were collected, the biofilm was removed, and bacteria were counted using 4′,6-diamidino-2-phenylindole (DAPI) staining or fluorescence in situ hybridization (FISH) with probes targeted to Domain Bacteria and to the α-, β-, and γ-Proteobacteria. DAPI-stained cells and Bacteria were more abundant in 1999 than 1998. Differences in abundance between years may have been related to water velocity, which was much lower in 1999 than 1998. The α-Proteobacteria were the most abundant group in the biofilm (up to 40% of Domain Bacteria), whereas the γ-Proteobacteria were the least abundant (generally <10%). Percentages of β-Proteobacteria were much greater in 1999 than 1998 (26% and 15%, respectively). Abundances of biofilm α- and β-Proteobacteria were much greater on exposed leaves than on dialysis-bag-enclosed leaves, suggesting that large numbers of cells may colonize leaf surfaces from stream water. In conclusion, Proteobacteria made up most bacteria on the surface of decomposing maple leaves. Erosion of cells from the biofilm and colonization from stream water may have been important factors controlling biofilm development.