In aquatic ecosystems, the dynamic processes of microbial communities are largely driven by heterotrophic microorganisms that rely on organic compounds for their carbon needs. This study was designed to provide a comprehensive assessment of the functional capabilities of these heterotrophic microbial communities residing in the surface waters and underlying sediments of both lake and riverine environments. Sediment and surface water samples were collected from six locations: three river sites (SC1, SC2, SC3) and three lake sites (LC1, LC2, LC3). The Biolog EcoPlate™ method was employed to profile the carbon utilization of microbial communities, and samples were analyzed for various physicochemical properties including moisture content, pH, electrical conductivity, turbidity, particle size distribution, heavy metals content, and major ions in water. Statistical analyses, such as unpaired two-sample t-tests, paired comparisons, and analysis of variance (ANOVA), were conducted to determine differences between sample groups, with post hoc Tukey’s Honest Significant Difference (HSD) tests and cluster analysis used to identify specific differences and natural groupings within the data. The results revealed that environmental conditions varied across the sites, with lake sites generally showing higher air and water temperatures and lower turbidity compared to river sites, while dissolved oxygen levels were consistently high across all sites. In terms of carbon utilization, site LC1 exhibited the highest utilization among lake sediments, utilizing 28 of the 31 substrates, while SC2 demonstrated the highest utilization among river sediments, using all 31 substrates. Surface water samples from LC1, SC1, and SC2 displayed higher microbial activity compared to LC2, LC3, and SC3. River samples showed significantly higher utilization of specific carbon sources such as phenylethylamine, L-threonine, D-xylose, and D-malic acid compared to lake samples, and sediment samples exhibited greater overall carbon utilization than surface water samples (P < 0.00001). Cluster analysis indicated that sediment microbial communities showed higher similarity between sites compared to surface water communities, suggesting more stable metabolic adaptations in sediment environments. The study highlights significant differences in microbial carbon utilization between lake and river environments, with river sediments showing more diverse metabolic capabilities, underscoring the critical influence of substrate type and environmental conditions on microbial community structure and function. In summary, the Biolog Ecoplate analysis emerged as a valuable method to differentiate the metabolic diversity of microbial communities across different aquatic habitats. By revealing distinct utilization patterns, this study enhances our understanding of how sediments support a broader and more stable metabolic network compared to surface waters, and how these patterns are consistent across lake and river environments. Such insights are foundational for further ecological research and environmental management strategies.