Isavuconazole (BAL4815) is a promising novel broad-spectrum triazole in late-stage clinical development that has proven active in vitro against Aspergillus and Candida species. We compared the in vitro activities of this agent with those of voriconazole and fluconazole by the CLSI (formerly NCCLS) M38-A and M27-A2 procedures against a large collection of 1,007 relevant opportunistic fungi collected from 1986 to 2007: Aspergillus spp. (n = 702), Candida spp. (n = 218), Zygomycetes (n = 45), Scedosporium spp. (n = 22), and Fusarium spp. (n = 20). All Candida isolates were from patients with candidemia. For isavuconazole, these techniques were also compared with the Etest. Isavuconazole and voriconazole had MICs at which 50% and 90% of isolates were inhibited (MIC50 and MIC90), respectively, of 1 and 1 microg/ml and 0.5 and 1 microg/ml against Aspergillus spp. and of < or = 0.015 [corrected] and 0.03 microg/ml and 0.25 and 0.125 microg/ml against Candida spp. (including fluconazole-resistant strains). The MIC50 partial and complete inhibition end points of isavuconazole and voriconazole against the non-Aspergillus molds were as follows: 1 and 2 microg/ml and 16 and >16 mug/ml against Zygomycetes; 1 and 4 microg/ml and 0.25 and 0.5 microg/ml against Scedosporium apiospermum; 4 to 16 and >16 microg/ml and 4 to 8 and 16 to >16 microg/ml (ranges) against Scedosporium prolificans; and 16 and 16 microg/ml and 4 and 4 microg/ml against Fusarium spp. Isavuconazole showed minimal fungicidal concentrations for 50% and 90% of the isolates of 1 and 1 microg/ml against Aspergillus, 16 and >16 microg/ml against Candida, and 4 and >16 microg/ml against Zygomycetes, respectively, and >16 microg/ml against the remaining molds. The Etest proved to be a suitable alternative method for determining the antifungal activities of isavuconazole against Aspergillus and Candida; the Etest results showed 96% and 93% agreement with the results of the CLSI M38-A and M27-A2 methods, respectively.