Iron acquisition is crucial for the growth of Aspergillus fumigatus. A. fumigatus biofilm formation occurs in vitro and in vivo and is associated with physiological changes. In this study, we assessed the effects of Fe chelators on biofilm formation and development. Deferiprone (DFP), deferasirox (DFS), and deferoxamine (DFM) were tested for MIC against a reference isolate via a broth macrodilution method. The metabolic effects (assessed by XTT [2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt]) on biofilm formation by conidia were studied upon exposure to DFP, DFM, DFP plus FeCl3, or FeCl3 alone. A preformed biofilm was exposed to DFP with or without FeCl3. The DFP and DFS MIC50 against planktonic A. fumigatus was 1,250 mu M, and XTT gave the same result. DFM showed no planktonic inhibition at concentrations of <= 2,500 mu M. By XTT testing, DFM concentrations of < 1,250 mu M had no effect, whereas 2,500 mu M increased biofilms forming in A. fumigatus or preformed biofilms (P < 0.01). DFP at 156 to 2,500 mu M inhibited biofilm formation (P < 0.01 to 0.001) in a dose-responsive manner. Biofilm formation with 625 mu M DFP plus any concentration of FeCl3 was lower than that in the controls (P < 0.05 to 0.001). FeCl3 at >= 625 mu M reversed the DFP inhibitory effect (P < 0.05 to 0.01), but the reversal was incomplete compared to the controls (P < 0.05 to 0.01). For preformed biofilms, DFP in the range of > 625 to 1,250 mu M was inhibitory compared to the controls (P < 0.01 to 0.001). FeCl3 at >= 625 mu M overcame inhibition by >= 625 mu M DFP (P < 0.001). FeCl3 alone at >= 156 mu M stimulated biofilm formation (P < 0.05 to 0.001). Preformed A. fumigatus biofilm increased with 2,500 mu M FeCl3 only (P < 0.05). In a strain survey, various susceptibilities of biofilms of A. fumigatus clinical isolates to DFP were noted. In conclusion, iron stimulates biofilm formation and preformed biofilms. Chelators can inhibit or enhance biofilms. Chelation may be a potential therapy for A. fumigatus, but we show here that chelators must be chosen carefully. Individual isolate susceptibility assessments may be needed.