We compared the in vitro pharmacodynamics of amphotericin B, itraconazole, and voriconazole against Aspergillus, Fusarium, and Scedosporium species with a combination of two non-culture-based techniques: the tetrazolium salt 2,3-bis-(2-methoxy-4-nitro-5-[(sulfenylamino)carbonyl]-2H-tetrazolium-hydroxide) (XTT) colorimetric reduction assay, and fluorescent microscopy with the cellular morbidity dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC) to directly visualize hyphal damage. Amphotericin B exhibited species-specific concentration-dependent activity, with 50% effective concentrations (EC(50)s) ranging from 0.10 to 0.12 mg/ml for A. fumigatus, 0.36 to 0.53 mg/ml for A. terreus, 0.27 to > or = 32 mg/ml for F. solani, 0.41 to 0.55 mg/ml for F. oxysporum, and 0.97 and 0.65 mg/ml for S. apiospermum and S. prolificans, respectively. Similarly, itraconazole inhibited the growth of A. fumigatus and A. terreus isolates with MICs of <1 mg/ml (EC(50) 0.03 to 0.85 mg/ml) and S. apiospermum, but was not active against Fusarium species or S. prolificans. Voriconazole effectively inhibited the growth of Aspergillus, Fusarium, and S. apiospermum (EC(50) 0.10 to 3.3 mg/ml) but had minimal activity against a multidrug-resistant isolate of F. solani or S. prolificans. Hyphal damage visualized by DiBAC staining was observed more frequently with voriconazole and amphotericin B versus itraconazole. These data highlight the species-specific differences in antifungal pharmacodynamics between mold-active agents that could be relevant for the development of in vitro susceptibility breakpoints and antifungal dosing in vivo.