Abstract: Microbial natural products are valuable resources for developing effective and safe herbicides. In this study, 70 strains of actinomycetes were selected for investigation. Initially, strains with strong herbicidal activity were identified through seed germination inhibition tests using Arabidopsis thaliana. The active herbicidal substances were then tracked, separated, and purified through solvent extraction and various chromatographic techniques, including normal silica gel, reverse silica gel, dextran gel, and other media. This was combined with activity testing and structural analysis based on nuclear magnetic resonance（NMR）spectroscopy data. The results indicate that one strain of Rhodococcus ruber was identified, showing significant inhibitory activity on Arabidopsis seed germination. From its fermentation products, a compound designated as compound 1 was isolated and identified as phenylacetic acid based on NMR data. Herbicidal activity assays revealed that phenylacetic acid achieved a 90% inhibition rate of Arabidopsis seed germination at a concentration of 60 µg·mL⁻¹, with a dose-dependent test yielding an IC₅₀ value of 36.07 µg·mL⁻¹. The research results reveal the potential application of Rhodococcus ruber and its metabolite phenylacetic acid in the development of natural herbicides.