Multiple forces structure natural microbial communities, but the relative roles and interactions of these drivers are poorly understood. Gradients of physical and chemical parameters can be especially influential. In traditional ecological theory, variability in environmental conditions across space and time represents habitat heterogeneity, which may shape communities. Here we used aquatic microbial communities as a model to investigate the relationship between habitat heterogeneity and community composition and dynamics. We defined spatial habitat heterogeneity as vertical temperature and dissolved oxygen (DO) gradients in the water column, and temporal habitat heterogeneity as variation throughout the open-water season in these environmental parameters. Seasonal lake mixing events contribute to temporal habitat heterogeneity by destroying and re-creating these gradients. Because of this, we selected three lakes along a range of annual mixing frequency (polymictic, dimictic, meromictic) for our study. We found that bacterial community composition (BCC) was distinct between the epilimnion and hypolimnion within stratified lakes, and also more variable within the epilimnia through time. We found stark differences in patterns of epilimnion and hypolimnion dynamics over time and across lakes, suggesting that specific drivers have distinct relative importance for each community.