Freshwater bacteria play key roles in biogeochemical cycling and contribute significantly to biomass and energy fluxes. However, studies of Great Lakes ecosystem dynamics often omit bacteria. Here, we used high throughput sequencing to analyze how bacterial diversity and community composition (BCC) vary seasonally along the long-term Muskegon estuary to pelagic research transect. Diversity was higher in the estuary than Lake Michigan, in spring compared to summer, and for particle-associated (PA) relative to free-living (FL) fractions. PA communities were distinct from and more variable than FL communities. For both fractions, spring BCC was more similar between estuary and nearshore Lake Michigan compared to offshore waters. In summer and fall, nearshore and offshore BCC were more similar compared to estuary BCC. Most abundant taxa were inferred to be chemoorganoheterotrophs. While, as a whole, this functional group only showed habitat preference for the PA fraction, we observed phylum- and class-level seasonal and spatial preferences. Chemoorganoheterotrophs that also perform bacteriorhodopsin-mediated phototrophy, such as acI Actinobacteria and LD12, strongly preferred FL fractions. Photoautotrophs (Cyanobacteria) were least abundant in spring, when mixotrophic methylotrophs were more abundant, particularly in the estuary. Organisms with chemolithotrophic capabilities, including a mixotrophic, highly abundant Limnohabitans (Lhab-A1) OTU, showed limited spatiotemporal patterns. One exception was Nitrosospira, an autotrophic ammonium oxidizer, which peaked in deep offshore waters in fall. Nitrosospira co-occurred with Chloroflexi CL500-11, which likely mineralizes nitrogen-rich organic matter in deep waters. These spatiotemporal BCC shifts suggest differences in bacterially mediated elemental cycling along estuary to pelagic gradients in Lake Michigan.