Phytopathogenic fungi would induce a variety of plant diseases, resulting in a severe reduction of agricultural output. However, the current plant disease control is mainly dependent on the environmentally and healthily hazardous chemical fungicides. Thus, the present work aimed to isolate an effective antagonistic microorganism against various soilborne phytopathogenic fungi. By dual culture with Rhizoctonia solani, a novel Streptomyces specie, Streptomyces sp. N2, was screened out from a total of 167 isolated actinomycetes, which displayed a strong inhibitory effect on R. solani (26.85 +/- 1.35 mm of inhibition zone diameter). By means of macroporous resin and silica gel column chromatography coupled with preparative HPLC, an antifungal metabolite (3-methyl-3,5-amino-4-vinyl-2-pyrone, C6H7O2N) was isolated and purified from Streptomyces sp. N2. The bioassay results showed that the purified antifungal metabolite could not only possess a broad-spectrum inhibitory effect on a range of plant pathogenic fungi in vitro (e.g., R. solani, Pyricularia grisea, Fusarium oxysporum f. sp. niveum, F. oxysporum f. sp. vasinfectum, Penicillium italicum, and Colletotrichum gloeosporioides), but also had a significantly effective in vivo biocontrol efficacy on grape fruits anthracnose caused by C. gloeosporioides. Microscopic observation indicated that the antifungal metabolite from Streptomyces sp. N2 would exert its antimicrobial activity by disorganizing the cytoplasmic organelles of phytopathogenic fungi. The above results suggested that Streptomyces sp. N2 was one of promising fungicide for biocontrol of fungal plant diseases, especially due to its broad-spectrum and effective antagonist on various plant pathogens.