Abstract: The pesticide residues might have certain affection on the microorganisms during the food fermentation, while enzymes of the microorganisms play important roles in the whole process. This paper mainly focused on the inference of hexaconazole and myclobutanil enantiomers on the typical enzyme activities and protein content of yeast and Aspergillus oryzae. It was found that the enzyme activities were significantly different when treated with two different enantiomers. In detail, (+)-hexaconazole had certain inhibition effect on SOD activity, while the (-)-enantiomer exhibited activation effect. (+)-Myclobutanil exhibited less inhibition effect on the yeast lipase than (-)-myclobutanil at low concentration, while at high concentration, those two enantiomers showed different behavior on the enzyme activity. (+)-Hexaconazole was also found to promote the lipase of Aspergillus oryzae, while the enzyme was inhibited by (-)-enantiomer. In summary, it was confirmed that the enantiomers of chiral pesticides exhibited different behaviors on the enzyme activities of the microorganisms. Furthermore, the wine component and safety might be affected by the pesticide residues.