Enantioselectivity of chiral pesticide enantiomers should be taken into consideration in pesticide application and environmental risk assessment. The phytotoxicity of the enantiomers of napropamide to cucumber, soybean, and the bioactivity to the target weeds Poa annua and Festuca arundinacea have been studied in this work. To the nontarget crops, the influences of napropamide on the root, shoot, fresh weight, chlorophyll, superoxide dismutase (SOD) and catalase (CAT) activities and membrane lipid peroxides have been studied. (-)-Napropamide was more toxic than the racemate and (+)-napropamide to soybean and cucumber in terms of root, shoot and fresh weight. The content of chlorophyll was not affected by napropamide. The impacts on the activities of SOD, CAT and membrane lipid peroxides showed that napropamide could induce the oxidative stress and rac-napropamide caused a stronger oxidative damage to cucumber and soybean than (-)-napropamide and (+)-napropamide. For the target weeds, the influences of napropamide on root, shoot and fresh weight have been studied. (-)-Napropamid was more active to P. annua, while rac-napropamide was more active to F. arundinacea. To reduce environmental pollution and improve the effectiveness of chiral pesticide, single enantiomer should be developed and produced. This work may provide evidence for developing optical pure product.