Heterotrophic bacteria, total coliforms, and Escherichia coli in naturally developed riverbed biofilms were investigated. Water samples and biofilms developed on surface of pebbles were collected from five stations located in pristine streams to an urban stretch in the Yamatogawa watershed, Japan. Sampling was done six times from February to October in 2007. Indicator bacteria generally indicated increasing trends towards downstream in streambed biofilm as well as in water column. Biofilm generally contained heterotrophic bacteria in greater densities than the river water, while indicating a significant positive correlation(r = 0.65, P <0.01). Although the correlation of total coliforms in water and biofilm was significant (r = 0.60, P < 0.01), the relation was not apparent. Correlation of E. coli in biofilm and in water was also significant (r = 0.50, P <0.01), but the increase of E. coli towards downstream to the urbanized area was greater in water column than in biofilm. Therefore, it was considered that the attachment of planktonic E. coli to biofilm was considered negligible in the river environment. The positive thermal effect of water temperature suggests the growth of E. coli in the biofilm. Detection of E. coli in biofilm may not directly mean the presence of fecal contamination in the river. In the river water quality assessment, riverbed biofilms can serve as a source of indicator bacteria causing a false-positive result especially in the pristine areas.