We report here the first evaluation of isavuconazole for inhibition of A. fumigatus CYP51 and of sterol biosynthesis in the fungus. Voriconazole and isavuconazole both bound tightly to recombinant AfCYP51A and AfCYP51B isolated in E. coli membranes. CYP51 reconstitution assays confirmed AfCYP51A and AfCYP51B in addition to three AfCYP51A mutants (G54W, L98H and M220K) were strongly inhibited by both triazoles. Voriconazole bound relatively weakly to purified HsCYP51 unlike isavuconazole, which bound tightly. However, isavuconazole was a relatively poor inhibitor of HsCYP51 activity with an IC50 value of 25 μM which was 55- to 120-fold greater than those observed for the A. fumigatus CYP51 enzymes, albeit not as poor an inhibitor of HsCYP51 as voriconazole which gave an IC50 value of 112 μM. Sterol analysis of triazole-treated A. fumigatus Af293 cells confirmed isavuconazole and voriconazole both inhibited cellular CYP51 activity with the accumulation of 14-methylated sterol substrates and depletion of ergosterol levels. Isavuconazole elicited a stronger perturbation of the sterol composition in Af293 than voriconazole at 0.0125 μg ml-1 indicating increased potency. However, complementation studies in Saccharomyces cerevisiae using strains containing AfCYP51A and AfCYP51B indicated isavuconazole to be equally as effective at inhibiting CYP51 activity as voriconazole. These in vitro studies suggest isavuconazole is an effective alternative to voriconazole as an antifungal agent against the target CYP51 in Aspergillus fumigatus.