<jats:title>Abstract</jats:title><jats:p>A number of bacterial strains were isolated from the internal tissue of <jats:italic>Trapa japonica</jats:italic>. Of these, strain KPE62302H, which had a 16S rDNA sequence identical to that of <jats:italic>Streptomyces miharaensis</jats:italic> showed antifungal activity against several plant pathogens. Treatment of seeds with strain KPE62302H induced a significant reduction in the incidence of Fusarium wilt in tomato plants compared with untreated controls. An antifungal substance (FP‐1) was purified from the culture extract of strain KPE62302H using C18 flash and Sephadex LH‐20 column chromatography and reverse phase HPLC. Extensive spectrometric analysis using MS and NMR identified this as filipin III. FP‐1 inhibited the mycelial growth of plant pathogenic fungi such as <jats:italic>Alternaria mali, Aspergillus niger, Colletotrichum gloeosporioides, C. orbiculare, Cylindrocarpon destructans, Diaporthe citiri, Fusarium oxysporum</jats:italic> at 1–10 μg ml<jats:sup>–1</jats:sup> and also markedly inhibited the development of Fusarium wilt caused by <jats:italic>F. oxysporum</jats:italic> f.sp.<jats:italic> lycopersici</jats:italic> in tomato plants by treatment with 10 μg ml<jats:sup>–1</jats:sup> under greenhouse conditions. The efficacy of FP‐1 against Fusarium wilt was comparable to that of the synthetic fungicide benomyl. An <jats:italic>egfp</jats:italic> ‐tagged strain of KPE62302H confirmed its ability to colonize tomato plants. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)