To investigate the in vitro activity of olorofim (F901318), a novel broad-spectrum antifungal agent, against 150 strains belonging to 16 different cryptic species of Aspergillus by EUCAST and CLSI methodologies.Olorofim, amphotericin B, micafungin, posaconazole and voriconazole were tested against cryptic species belonging to Aspergillus fumigatus complex (n = 57), Aspergillus ustus complex (n = 25), Aspergillus niger complex (n = 20), Aspergillus flavus complex (n = 20), Aspergillus circumdati complex (n = 15) and Aspergillus terreus complex (n = 13) using EUCAST and CLSI methodologies for broth microdilution susceptibility testing of antifungal agents.Olorofim was the only drug with activity against all cryptic species of Aspergillus tested, including the multiresistant species Aspergillus lentulus, Aspergillus fumigatiaffinis and Aspergillus calidoustus. Geometric means of MICs for olorofim were lower (0.017, 0.015 and 0.098 mg/L, respectively, for EUCAST; and 0.015, 0.015 and 0.048 mg/L, respectively, for CLSI) than for amphotericin B (4.438, 12.699 and 0.554 mg/L, respectively, for EUCAST; and 0.758, 1.320 and 0.447 mg/L, respectively, for CLSI), voriconazole (2.549, 2.297 and 5.856 mg/L, respectively, for EUCAST; and 2.071, 1.741 and 5.657 mg/L, respectively, for CLSI) and posaconazole (0.307, 0.308 and 12.996 mg/L, respectively, for EUCAST; and 0.391, 0.215 and 9.514 mg/L, respectively, for CLSI).Olorofim shows encouraging in vitro activity against cryptic species of Aspergillus that can be hard to treat with current antifungal therapies. Further studies are warranted in order to assess its efficacy.