The spatial structures of chiral pesticide enantiomers can affect their activity, toxicity and behavior, thereby altering exposure risk. Identifying enantiomer differences and developing high-efficiency green enantiopure pesticide is an important strategy for reducing the negative effects of pesticides. In this study, after confirming the absolute configuration of pydiflumetofen enantiomers, fungicidal activity evaluation indicated that the activity of S-(+)-pydiflumetofen was 81.3-421 times higher than R-(-)-pydiflumetofen on three kinds of phytopathogens that control Fusarium wilt (Fusarium spp.), Alternaria rot (Alternaria alternata) and Southern blight (Sclerotinia rolfsii), which might be caused by the stronger binding ability of S-(+)-pydiflumetofen with the active site of the target protein. The coexistence of R-(-)-pydiflumetofen would enhance the toxicity of S-(+)-pydiflumetofen on zebrafish through synergistic effect. Low-activity R-(-)-pydiflumetofen was preferentially dissipated in soybean, soybean plants, cabbage and celery, which was opposite in soil. The persistence of S-(+)-pydiflumetofen in crops and degradability in soil were advantageous for pesticide effects and environmental protection. Based on the maximum residue limit (MRL) and hazard quotient (HQ), the dietary risks were determined to be acceptable for all crops. Thus, developing enantiopure S-(+)-pydiflumetofen products might be a high-efficiency and low-risk strategy, and more studies should be conducted in this aspect.