To examine spatial differences in bacterial communities along the Mahoning River in Northeast Ohio (USA), sediment samples were collected on two dates from three sites. Downstream portions (sites 2 and 3, in this study) of the Mahoning River have been highly impacted by human activities. Two approaches were used to characterize the bacterial community: fluorescent in situ hybridization (FISH) with taxon specific probes and denaturing gradient gel electrophoresis (DGGE). Bacterial numbers (per g ash free dry mass), based on staining with DAPI (4′, 6-diamidino-2-phenylindole) or FISH with Domain Bacteria, Acinetobacter calcoaceticus or Pseudomonas putida-specific probes, were higher at the most upstream site (site 1) compared to the more disturbed downstream sites (sites 2 and 3). In addition, the number of bands based on DGGE was higher at the upstream site (site 1) compared to the two downstream sites (2 and 3) during spring. However, in summer, the number of bands was similar among sites and the most upstream (1) and the middle site (2) had the same average number of bands. In spring, the percent similarity (based on the presence/absence of bands) among the three sites was relatively low compared to summer. In general, differences in the bacterial community were found among the sites with differing levels of anthropogenic disturbance but varied between the two dates examined.