In order to understand the impacts of the reservoir construction on the diversity and ecological network of different microbial communities, seven sampling sites were set up in the Hengshan Reservoir in 2021. Water samples were collected from the surface and bottom of the reservoir. After filtering and extracting total DNA samples, high-throughput sequencing was carried out based on 16S and 18S rDNA to investigate the response of community structure, molecular ecological network, and keystone species of different microbial groups to water environment changes. The results showed that the Richness, Simpson, Shannon, and Pielou's Evenness indices of bacterial community in the surface and bottom layers were higher than those in the eukaryote community. The dominant community of bacteria included Proteobacteria, Actinobacteria, and Bacteroidetes, and the eukaryote community included Arthropoda, Ciliophora, Ochrophyta, etc. Moreover, the density and average clustering coefficient of the microbial networks in the surface waters of different phytoplankton communities were higher than those in the bottom waters. It was also observed that the microbial ecological networks in the surface waters were more closely related, and the number of nodes and edges, as well as the number of keystone species, of bacterial communities in the surface and bottom layers were significantly higher than those in the eukaryote microbial communities, indicating that the bacterial community network was larger, and the cooperative relationship and network connectivity between species were stronger. The interaction between bacterial community and eukaryote community in different water depths was dominated by positive correlation, and the negative correlation of the two groups in the bottom layer was slightly greater than that in the surface, indicating that the competition between bottom-layer species was greater than that between surface-layer species. In addition, the environmental impact factors of all species and keystone species of the community in surface water were basically the same, but they differed greatly in deep water, indicating that the influence mechanism of water depth change on keystone species was not the same as that of all species. The results further revealed the effects of reservoir construction on the stability and interspecific interactions of different microbial communities and provided a theoretical basis for predicting variations in microbial community and material cycling in reservoirs.