Microorganism is an important part of freshwater ecosystem and its community composition is closely related to water environment. Five experimental groups were set up in the laboratory to simulate the population structure of the ecosystem by planting the evergreen submersible plant Vallisneria natans and introducing Bellamya aeruginosa, Hyriopsis cumingii and Carassius auratus. In this paper, the effects of different aquatic organisms on bacterial community structure were explored by constructing a multi-trophic freshwater ecosystem. The results showed that the dominant bacteria groups in the freshwater ecosystem were Proteobacteria, Actinobacteriota, Bacteroidota and Cyanobacteria. The existence of snails, mussels and fish greatly reduced the number of Cyanobacteria in the water, which could effectively control the overreproduction of Cyanobacteria. The bacterial Shannon index was higher in Vallisneria natans and Vallisneria natans-Bellamya aeruginosa-Hyriopsis cumingii, but lower in Vallisneria natans-Bellamya aeruginosa and Vallisneria natans-Bellamya aeruginosa-Hyriopsis cumingii-Carassius auratus. RDA results showed that there were significant differences in the abundance of microbial communities among different systems. The introduction of different aquatic organisms and the content of DO are the direct factors affecting the change of microbial community, and the change of nutrient concentration is mainly dependent on the interaction between aquatic organisms and microorganisms, which is an indirect factor affecting the change of microbial community. In conclusion, the construction of multi-trophic ecosystem can not only improve the water quality, but also increase the diversity of bacteria in the water, change the community structure of bacteria in the water, and make it more healthy and stable.