Chevalone B was isolated from the ethyl acetate extract of the marine sponge-associated fungus Aspergillus similanensis. The structure of Chevalone B was confirmed by 1D and 2D NMR spectroscopy. Chevalone B showed antimicrobial activity against Gram-positive and Gram-negative bacteria, Candida albicans, and multidr-ug-resistant strains from the environment. Studies on Chevalone B have shown its potential value in antimicrobial applications.