Cutinostatin B was purified from a culture filtrate of a strain No. 3112-4 of an actinomycete which had been isolated from a soil sample collected in Setagaya-ku, Tokyo, Japan. The structure of this compound was determined mainly by 2D NMR experiments to be N(α)-[N(α)-(2,3-dihydroxybenzoyl)-ornithyl]-N(δ)-hydroxyornithine-δ-lactum. Cutinostatin B inhibited the cutinase activity of Cladosporium fulvum when using β-naphthyl caproate as the substrate with an IC50 value of 28.9 μm (11.0 μg/ml), and inhibited 95% when using crude apple cutin as the substrate with a concentration of 263 μM (100 μg/ml).