Reactive oxygen species are a critical weapon in the killing of Aspergillus fumigatus by polymorphonuclear leukocytes (PMN), as demonstrated by severe aspergillosis in chronic granulomatous disease. In the present study, A. fumigatus-produced mycotoxins (fumagillin, gliotoxin [GT], and helvolic acid) are examined for their effects on the NADPH oxidase activity in human PMN. Of these mycotoxins, only GT significantly and stoichiometrically inhibits phorbol myristate acetate (PMA)-stimulated O₂⁻ generation, while the other two toxins are ineffective. The inhibition is dependent on the disulfide bridge of GT, which interferes with oxidase activation but not catalysis of the activated oxidase. Specifically, GT inhibits PMA-stimulated events: p47^phox phosphorylation, its incorporation into the cytoskeleton, and the membrane translocation of p67^phox, p47^phox, and p40^phox, which are crucial steps in the assembly of the active NADPH oxidase. Thus, damage to p47^phox phosphorylation is likely a key to inhibiting NADPH oxidase activation. GT does not inhibit the membrane translocation of Rac2. The inhibition of p47^phox phosphorylation is due to the defective membrane translocation of protein kinase C (PKC) βII rather than an effect of GT on PKC βII activity, suggesting a failure of PKC βII to associate with the substrate, p47^phox, on the membrane. These results suggest that A. fumigatus may confront PMN by inhibiting the assembly of the NADPH oxidase with its hyphal product, GT.