Pesticides are considered to be the second-largest non-point source pollution in water. Our research assayed the river network of typical agricultural areas in the middle and lower Yangtze River as the study area. Pesticides residues in aquatic environment were determined by QuEChERS, combined with high-performance liquid chromatography tandem mass spectrometry, or gas chromatograph-mass spectrometer. At chiral pesticides' levels, we detected pesticides contents in water, classified and counted the types of pesticides, and analyzed their environmental risk assessment. Furthermore, potential correlations between chiral pesticides concentrations and water quality indicators were assayed. Additionally, we explored their relations with microbial communities at species levels. Enantiomers of Diclofop-methyl, Ethiprole, Difenoconazole and Epoxiconazole were enantioselectively distributed. More interestingly, due to various chiral environment of the sampling site, the enantiomers of Tebuconazole Acetochlor, Glufosinate ammonium and Bifenthrin had completely different distributions at different sites. Based on that, the chiral pesticides Diclofop-methyl, Bifenthrin, Ethiprole, Tebuconazole and Difenoconazole are enantioselective to the risk of aquatic environment. Generally, enantiomeric selectivity had high positive correlations with total nitrogen and phosphorus. Then we found that chiral fate behavior of Tebuconazole and Paichongding in water might be affected by prokaryotes. In addition, the chiral behavior of Diclofop-methyl, Propiconazole, Difenoconazole, and Tebuconazole isomers in water might be negatively affected by eukaryotes. That research helped us to comprehensively understand the impact of non-point source pollution of chiral pesticides in aquatic environment and provided basic data support for developing biological and water quality indicators for monitoring pollution in aquatic environment.