In order to study the effect of Bacillus on eukaryotic microbial community structure and physical and chemical factors in grass carp ponds, high-throughput sequencing technology was used to analyze the eukaryotic microbial community structure in the experimental group (Bacillus added pond) and control group (ordinary pond). At the same time, the physical and chemical indicators of the two ponds were analyzed. The results showed that the contents of TN, NH₄⁺-N and NO₃^--N in the pond water of the experimental group were significantly lower than those in the control group in August and September(P<0.05). The results showed that the abundance of Cyclotella meneghiniana, Trichosporon, Chroomonas, Hedriocystis and Cyclotella in the pond water of the experimental group was significantly higher than that in the control pond(P<0.05). The Chao1 index and Shannon index of the eukaryotic microbes in the experimental group were significantly higher than those in the control group (P<0.05). The experimental results confirmed that by adding Bacillus to the pond, the structure of the eukaryotic microbial community in the water body could be changed, thereby realizing the adjustment of the physical and chemical factors of the pond. The research results could provide some theoretical support for reducing the pollution of tailwater tailwater to the water environment.