Bacterioplankton play a crucial role in mass cycling within reservoir ecosystems and drive biogeochemical cycles. Based on the vertical characteristics of water temperature and dissolved oxygen in Wan’an Reservoir, a deep-water reservoir in Southeastern China, this study utilized 16S rRNA gene amplicon sequencing technology to reveal variations in the composition, structure and assembly mechanism of bacterioplankton communities during different thermal stratification periods in the reservoir. The results indicated that the α-diversity of bacterioplankton was highest in the thermocline and lowest in the hypolimnion during the period of thermal stratification formation. During the stabilization period, α-diversity was higher in the hypolimnion and lower in both the thermocline and the mixed layer. In the recession period, α-diversity was elevated in both the thermocline and the hypolimnion, while it remained lower in the mixed layer. Molecular ecological network analysis indicated that during the recession periods of thermal stratification, bacterioplankton exhibited stronger positive correlations, lower modularity, and higher network complexity. During the period of thermal stratification formation, the proportion of key species was highest (30.00%), followed by the recession period (24.38%) and the stabilization period (21.60%). The neutral model explained 79.7%, 78.8%, and 64.1% of the community variations during the formation, stabilization, and recession periods, respectively. The bacterioplankton community assembly was primarily driven by stochastic processes; however, environmental factors also significantly influenced bacterioplankton community structure. Throughout the three periods of thermal stratification, the bacterioplankton community was mainly affected by dissolved oxygen, chlorophyll-a, total nitrogen and total phosphorus. This study aids in the management of water environment and ecological status in reservoirs and the assessment of the health of aquatic ecosystems.