[Objective] Endophytic actinomycete FRo2 with activity against agricultural pathogenic fungi was isolated from the roots of Dongxiang wild rice (Oryza rufipogon). We identified FRo2 and separated its antimicrobial substances. [Methods] Morphological, physiology and biochemistry characteristics, chemotaxonomy analysis and 16S rRNA gene sequences were used to identify FRo2. The bioactivities were determined by tube plate method and mycelium growth inhibition method. According to nuclear magnetic resonance spectroscopy (NMR) extrapolation result, one antimicro-bial substances in the metabolites of FRo2 through normal-phase silica gel column chromatography and gel (Sephadex LH-20) column chromatography with bioassay-guided fractionation was isolated. [Results] FRo2 belonged to Streptomyces sp. and was similar to Streptomyces rochei. The metabo-lites had high inhibitory activities against Fusarium graminearumt, Rhizoctonia solani and other five agricultural pathogenic fungi, and the antimicrobial substances AW2 was elucidated as Dibutyl phthalate. [Conclusion] The material basis of the antibacterial constituents in antagonistic actinomy-cetes strain FRo2 was further clarified, and strain FRo2 is deserved to develop an biocontrol.