To investigate the effects of biofloc mode on the water environment and intestinal microbial community structure of largemouth bass, a 60-day culture experiment was conducted without water replacement in 300-L glass tanks. The experiment included a control group and a biofloc group, each with three replicates. The results showed the following: (i) the richness and diversity of the water environment and fish intestinal microbial community increased under the biofloc model; (ii) Proteobacteria, Patescibacteria, and Bacteroidota were the dominant phyla in the water environment of largemouth bass, while Proteobacteria, Firmicutes, Bacteroidota, Patescibacteria, and Actinobacteriota were the dominant phyla in the gut of largemouth bass. However, differences in the relative abundance and community structure of microorganisms were observed between the two groups, suggesting that the biofloc system impacts both the water environment and intestinal microbial community structure in largemouth bass culture. (iii) A correlation analysis between water quality indices and enzyme activity with microbial abundance revealed that microbial community composition could effectively reflect water quality and fish physiological health. Based on the analysis of microbial community structure, this study offers a theoretical foundation for integrating largemouth bass culture with the biofloc system, and provides valuable data for future health management and water quality control in largemouth bass production.