This study was conducted to elucidate microbiological characteristics of river water and groundwater communities in order to improve our conceptual and predictive understanding of river and groundwater ecosystem processes, functioning and management. Rouge River bacterial communities from shallow groundwater and river water were screened using Biolog Ecoplates, which test for oxidation of selected carbon sources and by culturing heterotrophic bacteria. The isolates cultured from the samples were also characterized using the 16SrRNA gene-based approach. The patterns of utilization of the groups of carbon substrates by the microbial communities revealed differences between river water and groundwater samples. Carbohydrates, polymers, carboxylic acids and amino acids were highly utilized by the microbial communities in the river samples, while carbohydrates, polymers, amino acids and phenolic compounds were metabolized in the groundwater samples. Sequence comparison results showed that the most prevalent phylum in all sites was the Firmicutes (low G+C, mostly gram-positive bacteria). The dominant isolates from this phylum were similar to Bacillus spp., (98% nucleotide identity), which represented approximately 62% of the total number of unique isolates. Also prevalent were the gamma-Proteobacteria, which were dominated by 16S rRNA sequences 98-99% similar to that of Pseudomonas spp. The observed profile of carbon sources metabolized reflected the catabolic potential of the river water and groundwater community. Many of the isolates recovered have been known to metabolize several organic substrates, and may have potential use in remediation organic contaminants from the Rouge River. Direct incubation water samples in Biolog Ecoplates produced patterns of metabolic response useful in the classification and characterization of river water and groundwater microbial communities. Heterotrophic bacteria isolated from the sites may play important roles in the fate of many organic and inorganic contaminants from the Rouge River, although future studies are needed to understand their response to these contaminants.