Pseudomonas aeruginosa and Aspergillus fumigatus are common opportunistic bacterial and fungal pathogens, respectively. They often coexist in airways of immunocompromised patients and individuals with cystic fibrosis, where they form biofilms and cause acute and chronic illnesses. Hence, the interactions between them have long been of interest and it is known that P. aeruginosa can inhibit A. fumigatus in vitro. We have approached the definition of the inhibitory P. aeruginosa molecules by studying 24 P. aeruginosa mutants with various virulence genes deleted for the ability to inhibit A. fumigatus biofilms. The ability of P. aeruginosa cells or their extracellular products produced during planktonic or biofilm growth to affect A. fumigatus biofilm metabolism or planktonic A. fumigatus growth was studied in agar and liquid assays using conidia or hyphae. Four mutants, the pvdD pchE, pvdD, lasR rhiR, and fasR mutants, were shown to be defective in various assays. This suggested the P. aeruginosa siderophore pyoverdine as the key inhibitory molecule, although additional quorum sensing-regulated factors likely contribute to the deficiency of the latter two mutants. Studies of pure pyoverdine substantiated these conclusions and included the restoration of inhibition by the pyoverdine deletion mutants. A correlation between the concentration of pyoverdine produced and anti fungal activity was also observed in clinical P. aeruginosa isolates derived from lungs of cystic fibrosis patients. The key inhibitory mechanism of pyoverdine was chelation of iron and denial of iron to A. fumigatus.