The EUCAST EDef 9.3.2 procedure recommends visual readings of azole and amphotericin B MICs against spp. Visual determination of MICs may be challenging. In this work, we aim to obtain and compare visual and spectrophotometric MIC readings of azoles and amphotericin B against isolates. A total of 847 isolates (  = 828 and cryptic species  = 19) were tested against amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole using the EUCAST EDef 9.3.2 procedure. Isolates were classified as susceptible or resistant/non-wild type according to the 2020 updated breakpoints. The area of technical uncertainty for the azoles was defined in the updated breakpoints. Visual and spectrophotometric (fungal growth reduction of >95% compared to the control, read at 540 nm) MICs were compared. Essential (±1 2-fold dilution) and categorical agreements were calculated. Overall, high essential (97.1%) and categorical (99.6%) agreements were found. We obtained 100% categorical agreements for amphotericin B, itraconazole, and posaconazole, and consequently, no errors were found. Categorical agreements were 98.7 and 99.3% for voriconazole and isavuconazole, respectively. Most of the misclassifications for voriconazole and isavuconazole were found to be associated with MIC results falling either in the area of technical uncertainty or within one 2-fold dilution above the breakpoint. The resistance rate was slightly lower when the MICs were obtained by spectrophotometric readings. However, all relevant mutants were correctly classified as resistant. Spectrophotometric determination of azole and amphotericin B MICs against isolates may be a convenient alternative to visual endpoint readings.