Microorganisms play pivotal roles within aquatic ecosystems, affecting their structure, functioning and services. However, little is known about the effects of water stratification and mixing on the aquatic microbial community dynamics in subtropical reservoirs. In this study, we explored vertical and seasonal patterns of microbial diversity in the Dongzhen Reservoir (southeast China). Quantitative PCR, quantitative RT-PCR, and 454 pyrosequencing were used for an in-depth characterization of the bacterial community across time (every three months for one year) and space (five different water depths). Our results indicated that thermal and oxygen stratification shaped the phylogenetic composition of microbial communities in the reservoir. There were significant differences in physical, chemical and microbiological parameters between epilimnion and hypolimnion (P < 0.05). The RNA: DNA ratios were significantly lower in epilimnion and metalimnion but rapidly increased in hypolimnion (P < 0.05), suggesting that microorganisms were more active at low temperatures, low dissolved oxygen concentrations and high TN/TP ratios. Redundancy analysis and pathway analysis revealed a complex interplay of various environmental and biological factors by explaining the spatiotemporal variations in bacterial communities. Adaptive reservoir management strategies should consider carefully the effects of water stratification and mixing, together with the distribution patterns of aquatic microorganisms.