AbstractBACKGROUNDPlant viral diseases are difficult to control and have caused serious damage to the agricultural industry. Recently, botanical biopesticides characterized by environment friendly, safe to non‐target organism and not as susceptible to produce drug resistance, have exhibited great potential to be developed as antiviral agents. To screen the natural products with antiviral effect, three alkaloids possessed anti‐tobacco mosaic virus (TMV) activity were isolated from Chelidonium majus and the modes of action were investigated.RESULTThe anti‐TMV effect of crude extracts at 10 mg mL−1 was 51.73%. Bioassay‐guided fractionation and isolation of the compounds with anti‐TMV activity were performed on the methanol extract of C. majus yielding three bioactive alkaloids namely: chelerythrine (1), chelidonine (2), and sanguinarine (3). The results of bioassay showed that chelerythrine exhibited great inactivation, proliferation inhibition and protection effects against TMV at 0.5 mg mL−1 with the efficiency of 72.67%, 77.52% and 59.34%, respectively. Chelidonine at 0.1 mg mL−1 can provide 54.90% and 64.45% inhibitions on TMV through inducing resistance in two kinds of tobacco. Sanguinarine showed a weaker protection for resisting TMV in comparison to chelerythrine and chelidonine.CONCLUSIONChelerythrine and chelidonine displayed significant inhibitions on TMV with different modes of action. These results provided important evidence that the extracts in C. majus might be a potential source of new drugs in controlling virus disease agriculturally.