We compared posaconazole M27-A2 and M38-A MICs to Etest and YeastOne MICs for 92 zygomycetes, 126 Aspergillus isolates, 110 Candida isolates, and Cryptococcus neoformans. Reference MICs were also correlated with inhibition zone diameters in millimeters (modified M44-A disk and Neo-Sensitabs tablet methods). Etest MICs were obtained on solidified (1.5% agar) RPMI 1640 (2% dextrose), and zone diameters were obtained on supplemented (2% glucose and 0.5 microg/ml methylene blue [for all isolates]) and nonsupplemented Mueller-Hinton (MH; molds only) agar. MICs and zone diameters were obtained between 16 and 72 h. The overall agreement (% MIC pairs within a three-dilution range) between reference posaconazole and YeastOne MICs was 98 to 100% at 16 to 24 h for zygomycetes and yeasts and 99% at 24 to 48 h for Aspergillus. The overall agreement was lower between reference posaconazole and Etest MICs (94 to 97%) and by both methods with amphotericin B for all species (95 to 99.3%). For yeasts, the correlation coefficient was similar between reference posaconazole MICs and either disk (R, 0.810) or tablet (R, 0.769) zone diameter at 24 h and was superior on MH agar for molds at 16 to 48 h (R, 0.804 and 0.799 for disk and tablet, respectively). For amphotericin B, the best correlation between reference MICs and zone diameters was observed at 16 to 48 h for molds on MH agar (R, 0.736 to 0.812 and 0.765 to 0.749 for disk and tablet, respectively) and at 48 h for yeasts (R, 0.681 and 0.503 for disk and tablet, respectively). These data suggest the potential value of these alternative broth dilution and agar diffusion methods for testing posaconazole and amphotericin B in the clinical laboratory against the species evaluated.