The susceptibility profile of 91 Sporothrix schenckii isolates in both growth phases was determined by microdilution test (Antifungal Susceptibility Testing of the European Committee for Antimicrobial Susceptibility Testing; AFST-EUCAST). Amphotericin B (AMB), itraconazole (ITC), posaconazole, ravuconazole and terbinafine were found active in vitro against both phases but minimum inhibitory concentrations values for mycelial phase were significantly higher. Fluconazole (FLC) and voriconazole (VRC) were inactive in vitro against both phases. The E-test technique was also performed with 41 representative isolates for AMB, FLC, ITC and VRC. Average agreement rates between yeast phase microdilution results and E-test results were high for AMB (77.5%) and FLC (87.8%), but low for ITC and VRC with rates of 56.4% and 54.5%, respectively. AFST-EUCAST is not the most recommended test to perform drug susceptibility testing of S. schenckii in clinical laboratories, and E-test could be an alternative methodology for this purpose, mainly when the activity in vitro of antifungal agents of AMB and FLC are evaluated.