Aims To evaluate the antimicrobial activities of an active compound isolated from the culture broth of Amphirosellinia nigrospora JS‐1675 against various plant pathogenic bacteria and fungi. Methods and Results While screening for bioactive secondary metabolites from endophytic fungi, we found that A. nigrospora JS‐1675 showed strong in vitro antibacterial activity against Ralstonia solanacearum. One compound (1) was isolated and identified as (4S, 5S, 6S)‐5,6‐epoxy‐4‐hydroxy‐3‐methoxy‐5‐methyl‐cyclohex‐2‐en‐1‐one. Growth of most of the tested phytopathogenic bacteria was inhibited by compound 1 and the ethyl acetate (EtOAc) layer except Pseudomonas syringae pv. lachrymans. Compound 1 also inhibited the mycelial growth of several plant pathogenic fungi. Both compound 1 and the EtOAc layer reduced bacterial leaf spot disease in detached peach leaves. They also suppressed the development of bacterial wilt on tomato seedlings quite effectively. Conclusions Amphirosellinia nigrospora JS‐1675 showed antimicrobial activity against plant pathogenic bacteria and fungi by producing compound 1. Significance and Impact of the Study This is the first report on the occurrence of compound 1 in A. nigrospora JS‐1675 and its efficacy against plant pathogenic bacteria and fungi. Their strong disease control efficacy against tomato bacterial wilt suggests that this fungus can be used as a microbial bactericide.