Marine biofouling is a common worldwide problem. Current toxic antifouling compounds cause serious harm to marine environment and human health. Natural nontoxic antifouling compound development has been the primary way to solve this problem. In an ongoing survey of sponge-associated bacteria to find an antidiatom adhesion property, one strain named UST050418-683 was isolated from 120 strains with the strongest activity inhibiting diatom adhesion and identified as Bacillus pumilus, based on the 16S rDNA sequence. The crude extract from the spent culture medium of this bacterium could inhibit diatom adhesion. The bioassay-guided fractionation was employed to isolate antifouling compounds. The ethyl acetate extract of the fermentation broth was isolated by multiple chromatograph techniques, (e.g. silica gel column chromatograph and reverse-phase decompression column chromatographic). The active fractions were further purified on the high-performance liquid chromatography equipped with a C18 column. The molecular structures were elucidated by the nuclear magnetic resonance spectroscopy and mass spectrometry, as well as gas chromatography analyses. These results indicated that fatty acids with an antidiatom adhesion property were isolated for the first time from a B. pumilus strain. It was suggested that fatty acids and other compounds from sponge-associated bacteria could be used as potential antifoulants.