The human pathogenic fungus Aspergillus fumigatus establishes dual biofilm interactions in the lungs with the pathogenic bacterium Pseudomonas aeruginosa. Pyripyropene A is a potent inhibitor of mammalian acyl-CoA cholesterol acyltransferase. Deletion of pyr2, encoding a non-reducing polyketide synthase essential for pyripyropene biosynthesis, showed reduced A. fumigatus Δpyr2-P. aeruginosa biofilm growth, altered macrophage responses, and attenuated mouse virulence in a chemotherapeutic murine model. We identified pyripyropene as a novel player in the ecology and pathogenic interactions of this important human fungal pathogen.