A phytotoxin from Xanthomonas campestris pv. retroflexus was isolated using a chromatographer and HPLC, and the components were identified to be a mixture of minor molecular compounds including organic acids and cyclo-(proline-phenylalanine). The greenhouse cultivation test was used to determine the influence of the isolated fractions on the growth of target weed redroot pigweed (Amaranthus retroflexus L). The experimental results demonstrated that the cyclo-(Pro-Phe) had the weed inhibit activity obviously on dicotyledonous weed and the mixture with six organic acids showed stronger bioactivity. Further, greenhouse and field test were processed, and the test showed that the use of the toxin appeared to have the potential to be developed further as a bioherbicide system to control weedy grasses.