To investigate the impact of periodic fluctuations in the water level fluctuation zone on soil microbial community structure in the Three Gorges Reservoir, this study focuses on the water level fluctuation zones of Longtanping and Lanlingxi near the dam. Soil samples were collected from three elevation intervals: under 160m, between 160-170m, and above 170m during spring, summer, and autumn. The microbial community structure was analyzed using phospholipid fatty acids (PLFAs). The results indicated that microbial community distribution was significantly influenced by elevation and season, with the bacterial abundance accounts for 73.5%. Microbial mass and bacterial abundance showed a "unimodal" distribution pattern with the increase of altitude, the microbial biomass in the 160-170m elevation interval was 24.5% and 22.5% higher than that in the intervals below 160m and above 170m, respectively, while the bacterial abundance was 8.7% and 3.2% higher than that in the other two intervals, respectively. Seasonally, the changes in microbial biomass and bacterial abundance over time also showed a "unimodal" distribution pattern, and reached the highest in summer, and the abundance of fungi and actinomycetes was the highest in autumn. The results of the two-way analysis showed that elevation has a significant effect on various microbial groups, but the influence of season on the microbial community is more pronounced. In each region, both season and elevation affected microbial groups, and there was a significant interaction effect of season and elevation on fungi. Fungi were more adapted to the Longtanping area, which had a higher pH, while there was no significant difference in the abundance of actinomycetes across different regions and elevations. Redundancy analysis (RDA) indicated that the two principal components explained 42.85% of the microbial community, with the environmental factors affecting the microbial community structure being mainly soil organic carbon (SOC, 33.8%), total soil nitrogen (TN, 1.1%), and pH (8.2%). SOC and pH are key driving factors for the microbial community structure, with pH primarily driving changes in the microbial community in Longtanping area, and SOC mainly influencing the microbial community in the lower pH region of Lanlingxi. Correlation analysis between soil physicochemical factors and biological groups indicated a positive correlation between microbial biomass and bacterial abundance, both of which were positively correlated with the environmental factors SOC, TN, and pH, whereas fungal abundance was negatively correlated with bacteria and actinomycetes, respectively. This study reveals the impact patterns of fluctuating water levels and seasonal changes on microbial communities, providing an important theoretical basis for the ecological protection and restoration of the fluctuating zone in the Three Gorges Reservoir.