Echinochloa crus-galli is one of the most noxious weeds in the world and causes yield losses in a variety of different field crops. Napropamide and acetochlor are herbicides commonly employed to control this weed. Both compounds are chiral, with enantiomers displaying different activities. However, it is unclear how the enantiomers of these two chiral herbicides act on different tissues of E. crus-galli. The objective of this paper is to investigate the action mechanism of napropamide and acetochlor in the roots and shoots of E. crus-galli. R‑enantiomers were found to be more active than either the racemates or S-enantiomers on the weed. The content of chlorophyll was not significantly affected by treatment with either enantiomer. The impacts on the activity for the oxidative stress enzymes, except catalase (CAT), showed that both napropamide and acetochlor enantiomers could induce oxidative stress. Furthermore, R‑enantiomers caused greater oxidative damage. Enhanced glutathione-S-transferase (GST) activity and expression of GST genes suggested both EcGSTF1 and EcGSTZ1 were present in the roots and shoots, and this will be helpful for detoxification. The changes in both the roots and shoots revealed the two herbicides displayed tissue selectivity in E. crus-galli. These results enable a better understanding on the mechanism of action for napropamide and acetochlor enantiomers on different tissues, including the shoots and roots in E. crus-galli