[Objective] The aim of this study was to isolate and identify plant pathogenic fungal strains with good herbicidal activity and to isolate active metabolites, for the development of new biological herbicides.[Methods] Phytopathogenic fungi were isolated and purified by solid medium inoculation. The targeted strain was identified by morphological observation and 5.8S rDNA sequencing analysis. The active components were separated and purified after screening and tracing, and the structure of the active monomer compounds was determined by spectral analysis. The herbicidal activity of active metabolite and its safety to common crops were determined by Petri dish biological analysis.[Results] Tea pathogen CY-H had good herbicidal activity. The inhibition rates of fermentation broth on radical growth of Echinochloa crusgalli and Amaranthus retroflexus were 94.6% and 77.3% respectively. CY-H was identified as Diaporthe sp.. The single compound CY1 was isolated from CY-H and identified as cytosporaphenones C. At the concentration of 100 μg/mL, CY1 had a good herbicidal activity against the A. retroflexus, with the inhibition rates of 57.1%, and it had a good safety against Triticum aestivum and Brassica napus with the inhibition rate of about 20%.[Conclusion] The tea pathogen CY-H could be potentially developed as a microbial herbicide.