The in vitro susceptibilities of three hundred eighty-one isolates representing two classes, five orders, nine families, 30 genera, and 51 species of ascomycetous fungi to voriconazole, itraconazole, and amphotericin B were tested by using a modification of the National Committee for Clinical Laboratory Standards M27-A reference method. For those fungi of known phylogenetic relatedness, drug MICs were consistently low for isolates among all clades, except for members of the family Microascaceae. The highest MICs of all drugs tested were consistently for the Microascaceae, supporting the observation of fungal phylogeny and corresponding susceptibility to antifungal drugs. Itraconazole and voriconazole have a broad range of activity against phylogenetically similar agents of hyalohyphomycosis, phaeohyphomycosis, chromoblastomycosis, and mycetoma.