Microorganisms as the vital component of aquaculture ecosystems, were involved in nutrient cycling, water quality regulation, and the health of cultured species. In this study, aquatic water samples were collected monthly to reveal microbial community compositions, functions, and interactions in different habitats (pond and waterway) during the culture stages of the Chinese swimming crab Portunus trituberculatus in Shandong Province. Due to the semi-closed conditions with limited water exchange, the α diversities were lower in the polyculture pond. The relative abundances of the dominant phyla varied dramatically, and most biomarkers were identified in the polyculture pond. The β diversity of the microbial communities significantly differed among culture stages and habitats. Furthermore, the heterogeneity of prokaryotic microorganisms was greater than that of eukaryotic microorganisms. In the polyculture pond, the microbial community was sensitive to nutrient cycling, with abundant functions related to “photoautotrophy”, “nitrogen_respiration” and “nitrate_respiration”, corresponding to P. trituberculatus culture activities. The co-occurrence network of the microbial community in the polyculture pond tended to be looser than that in the waterway, with a lower number of taxa and correlations. In addition, these networks had nonrandom patterns and formed “small world” topological structures. The low-abundance genera with a higher level of betweenness centrality scores were the hubs or connectors in the networks and might play an essential role in microbial community composition. Furthermore, potential pathogens (Vibrio, Acholeplasma, Photobacterium, and Flavobacterium) deserve more attention during P. trituberculatus culture. This study will be useful for understanding microbial communities in polyculture pond systems and will provide a reference for the health aquaculture of P. trituberculatus.