Seasonal variation is one of the factors that has an important role in biodiversity changes. Investigating how the prokaryotic community composition respond to the temporal variation is vital to understand the ecosystem functions. In this study, in order to investigate the effect of seasonal variation on the prokaryotic community composition in Yan Yean drinking water reservoir (YYR), located in North Melbourne, VIC, samples were collected in three different seasons (Summer, Autumn, and Winter 2021). The prokaryotic community composition was performed using 16S rRNA gene metabarcoding. The non-chimeric sequence reads were classified taxonomically into 48 phyla, 106 classes, 252 orders, 427 families and 819 genera. The results of Shannon diversity index showed that the richness and diversity of bacterial communities in different seasons changed significantly (F2,96 = 24.93, p < 0.001), with the samples from Winter having the least richness and diversity. A Principal Coordinates Analysis (PCoA) was performed using the Weighted UniFrac distance matrix. According to PCoA, bacterial communities tended to cluster according to the seasonal variation. This clustering was supported by PERMANOVA, which suggested that 47% of variance between communities were explained by seasonal variation (p = 0.001). In general, the microbial composition was comparable to the microbial composition in other freshwater systems. Actinobacteriota was the most dominant phylum, with the maximum abundance was detected in Winter. That was followed by Bacteroidota, and Proteobacteria, with the later showing the least abundance in Winter. The most other sequences belonged to Verrucomicrobiota, Cyanobacteria, and Planctomycetota. The consistent presence of the three most abundant phyla, Actinobacteriota, Bacteroidota, and Proteobacteria, suggest that these phyla can play a key role in YYR ecosystem functioning. The predicted functional profiles of the microbial community will be determined by PICRUSt using the QIIME2 output files.