The isolation and structure determination of a new chlorinated benzophenone antibiotic, pestalone (1), is described. The new compound was produced by a cultured marine fungus only when a unicellular marine bacterium, strain CNJ-328, was co-cultured in the fungal fermentation. The fungus, isolated from the surface of the brown alga Rosenvingea sp. collected in the Bahamas Islands, was identified as an undescribed member of the genus Pestalotia. The structure of 1, initially assigned with only modest confidence by combined spectral and chemical data, was confirmed by single-crystal X-ray analysis. Pestalone (1) exhibits moderate in vitro cytotoxicity in the National Cancer Institute's 60 human tumor cell line screen (mean GI(50) = 6.0 microM). More importantly, pestalone shows potent antibiotic activity against methicillin-resistant Staphylococcus aureus (MIC = 37 ng/mL) and vancomycin-resistant Enterococcus faecium (MIC = 78 ng/mL), indicating that pestalone should be evaluated in advanced models of infectious disease.