To investigate the effects of addition of EM on the water quality and microbial community structure in polyculture of Eriocheir sinensis and Micropterus salmoides , one-month EM treatment experiment was conducted with period Ⅰ (April 10th), period Ⅱ (April 20th), period Ⅲ (April 30th), and period Ⅳ (May 10th) under the analysis of water indexes and microbiome structure. The 16S rRNA high-throughput sequencing results showed that the dominant phyla were Proteobacteria, Actinobacteriota, Firmicutes and Cyanobacteria during the whole experiment period. Meanwhile, the relative abundance of Cyanobacteria in the control group was significantly higher than that of the experimental group in period Ⅲ and Ⅳ. Compared with the control group, Limnohabitans (20.34%) was the most dominant at genus level in period Ⅰ, whereas Exiguobacterium occupied the most abundant position in other three periods (56.33%, 38.11%, 17.88%). However, Rhodoferax (10.37%), Exiguobacterium (47.67%), and Mycobacterium (36.01%, 42.27%) were the most abundant in each period of the control group. The Sobs and PD indexes of the experiment group were significantly higher than those of the control group in period Ⅲ and Ⅳ. Compared with the control group, the total nitrogen (TN) of the experiment group was significantly decreased during the whole monitoring periods, moreover, the content of ammonia nitrogen decreased significantly in period Ⅱ, Ⅲ and Ⅳ. Correlation analysis of environmental factors showed that total nitrogen had the greatest impact on the bacterial community, and Exiguobacterium as the dominant probiotics in the experimental group were positively correlated with TP and negatively correlated with TN, NH₄⁺-N, NO₃^--N, NO₂^--N. Studies have shown that the addition of EM could improve the water quality of aquaculture water, effectively inhibite Cyanobacteria, significantly optimize the bacterial structure, and had significant in-situ water remediation function. It may promote green and sustainable development of aquaculture.