In this first comprehensive study of Lake Winnipeg’s microbial communities, limnetic and littoral euphotic zones were examined during each season from 2016 through 2020. Classical cultivation and modern high-throughput sequencing techniques provided quantification and identification of key phototrophic populations, including aerobic anoxygenic phototrophs (AAP). Annual dynamics found total heterotrophs reached 4.23 × 106 CFU/g in littoral sands, and 7.69 × 104 CFU/mL in summer littoral waters on oligotrophic media, higher counts than for copiotrophic compositions. Limnetic numbers inversely dipped to 4.34 × 103 CFU/mL midsummer. Cultured AAP did not follow heterotrophic trends, instead peaking during the spring in both littoral and limnetic waters as 19.1 and 4.7% of total copiotrophs, or 3.9 and 4.9% of oligotrophs, decreasing till autumn each year. Complementary observations came from environmental 16S V4 rRNA gene analysis, as AAP made up 1.49 and 1.02% of the littoral and limnetic sequenced communities in the spring, declining with seasonal progression. Spatial and temporal fluctuations of microbes compared to environmental factors exposed photosynthetic populations to independently and regularly fluctuate in the ecosystem. Oxygenic phototrophic numbers expectantly matched the midsummer peak of Chl a and b, oxygenic photosynthesis related carbon fixation, and water temperature. Independently, AAP particularly colonized spring littoral areas more than limnetic, and directly corresponded to habitat conditions that specifically promoted growth: the requirement of light and organic material.