<i>Myrothecium verrucaria</i> infects various plants, but the mechanism of infection is unknown. We studied infection of using <i>M. verrucaria</i> strain MAFF840074, that causes Myrothecium leaf spot of mulberry, to mulberry leaves. Infection by conidia washed with water was low, and an extract of the fungal culture grown on potato sucrose agar caused necroses of the leaves. Infection by conidia was high and the diameter of the resulting lesions was larger if the extract was added to the conidia at the time of inoculation. Roridin A, verrucarins A and J, and an unidentified toxic substance (toxic substance A) were isolated from the extract based on induction of necroses on mulberry leaves using high performance liquid chromatography (HPLC). Roridin A and verrucarin J were more toxic to mulberry leaves than verrucarin A and the toxic substance A. The toxicity of each substance tended to be higher on sensitive mulberry cultivars than on cultivars resistant to the disease. Toxic substances with the same Rf value as roridin A, verrucarin A and the toxic substance A using thin layer chromatography were detected from extracts of mulberry leaves after inoculation with the fungus. With HPLC, the substances in the extract and concentrations of them were determined to be highest (roridin A: 5×10<sup>-7</sup>M, verrucarin A: 1.6×10<sup>-7</sup>M and the toxic substance A: 21×10<sup>-7</sup>M) at 12 days after the inoculation during the 14-d period. However, verrucarian J was not detected from the extract by the methods. Washed conidia of the fungus did not infect mulberry leaves (cv. Shin Ichinose) but infect the leaves when roridin A or verrucarin A at concentration that they caused necroces to the leaves or higher were added at the time of inoculation. Therefore, the results suggested that roridin A, verrucarin A and the toxic substance A might play some roles in the pathogenicity of the fungus.