Aspergillus flavus is the second most common species causing invasive aspergillosis after A. fumigatus. In certain countries like India, Sudan, and Saudi Arabia, A. flavus is most frequently isolated from patients with fungal rhinosinusitis and endophthalmitis. A. flavus exhibit an increased resistance to antifungal agents compared to A. fumigatus. We determined the in vitro activity of isavuconazole, voriconazole, posaconazole, itraconazole, amphotericin B, caspofungin, micafungin, and anidulafungin against 208 isolates of A. flavus by the EUCAST method and compared with the results obtained by the CLSI method. Isavuconazole and voriconazole MICs were =2 mug/mL in 99% and 95%, respectively. Posaconazole and itraconazole MICs were =0.5 and =1 mug/mL, respectively, for all isolates. MICs of amphotericin B were >/=2 mug/mL in 91%; 36% of them exhibited MICs of >/=8 mug/mL. All echinocandins demonstrated good anti-A. flavus activity. The essential agreement of the MIC/MEC results by EUCAST with CLSI broth dilution method assessed at +/-2 dilutions was good for itraconazole (97.8%), voriconazole (100%), posaconazole (98.3%), isavuconazole (98.9%), caspofungin (99.4%), and anidulafungin (100%), but poor for amphotericin B (53.5%) and micafungin (79.1%).