Recently isavuconazole, an experimental triazole agent, was found to be active against Aspergillus species. As Aspergillus flavus is the second-most common Aspergillus species isolated from human infection and the fungus has not been widely tested against the drug, we studied a large collection of clinical (n = 178) and environmental (n = 10) strains of A. flavus against isavuconazole and compared the results with seven other Aspergillus-active antifungal agents (some of them triazoles, others echinocandins or polyene antifungals: voriconazole, posaconazole, itraconazole, caspofungin, anidulafungin, micafungin and amphotericin B) using Clinical and Laboratory Standards Institute methods. Strains with high minimal inhibitory concentrations (MICs) were tested by E-test as well. The strains were collected from two different geographical locations (India and the Netherlands). Three isolates (1.6%) had high MIC (2 mg l−1 by microbroth dilution and 8 mg l−1 by E-test) for amphotericin B. Isavuconazole showed good activity against A. flavus strains with MIC50 and MIC90 values of 1 mg l−1. As compared with voriconazole (the drug recommended for primary therapy of aspergillosis), isavuconazole had better activity (99.5% of strains had MIC of ≤1 mg l−1 for isavuconazole, compared to 74% of strains with same MIC for voriconazole). All strains were, following recently proposed clinical breakpoints, susceptible for the triazoles tested except three strains, which had MICs of 4 mg l−1 for voriconazole. Testing these strains with high MIC by E-test, gave results of 0.5–2 mg l−1. Posaconazole had the lowest MIC50 and MIC90 of 0.125 mg l−1 and 0.25 mg l−1, respectively. Among echinocandins, 97% of strains had a minimum effective concentration (MEC) of ≤0.5 mg l−1 for caspofungin, and all strains had a MEC of ≤0.016 mg l−1 and ≤0.125 mg l−1 for anidulafungin and micafungin, respectively.