Microbial communities are vital to the ecology of the Laurentian Great Lakes, yet the influence of point sources, community assembly processes, and hydrodynamic circulation on their abundance and structure remains poorly understood. To address this, we analyzed 16S rDNA gene sequencing, flow cytometry, and water quality data from Lake Ontario during May and September of 2023. Communities were grouped into three depth-month categories: Shallow May, Shallow September, and Deep. Deep communities were dominated by drift, with some dispersal limitation, while shallow communities were shaped by homogenizing selection, especially in September, with contributions from drift. Actinobacteria were associated with homogenizing selection, Alphaproteobacteria with drift, and Bacteroidia in May and Chloroflexi in September with dispersal limitation. Upwellings altered microbial abundance and composition, modifying nitrogen cycling gene abundance. These findings provide insights into the ecological implications for Great Lakes health and conservation, emphasizing how upwelling events create unique microbial communities within the lake.