Information on the biodegradation potential of lake and river microbial communities is essential for watershed management. The water draining into the lake ecosystems often carries a significant amount of suspended sediments, which are transported by rivers and streams from the local drainage basin. The organic carbon processing in the sediments is executed by heterotrophic microbial communities, whose activities may vary spatially and temporally. Thus, to capture and apprehend some of these variabilities in the sediments, we sampled six sites: three from the Saint Clair River (SC1, SC2, and SC3) and three from Lake Saint Clair in the spring, summer, fall, and winter of 2016. Here, we investigated the shifts in metabolic profiles of sediment microbial communities, along Saint Clair River and Lake Saint Clair using Biolog EcoPlates, which test for the oxidation of 31 carbon sources. The number of utilized substrates was generally higher in the river sediments (upstream) than in the lake sediments (downstream), suggesting a shift in metabolic activities among microbial assemblages. Seasonal and site-specific differences were also found in the numbers of utilized substrates, which were similar in the summer and fall, and spring and winter. The sediment microbial communities in the summer and fall showed more versatile substrate utilization patterns than spring and winter communities. The functional fingerprint analyses clearly distinguish the sediment microbial communities from the lake sites (downstream more polluted sites), which showed a potential capacity to use more complex carbon substrates such as polymers. This study establishes a close linkage between physical and chemical properties (temperature and organic matter content) of lake and river sediments and associated microbial functional activities.