Objective The in vitro activity of isavuconazole was determined for 1677 Candida and 958 Aspergillus isolates from 2012 to 2014 with voriconazole as comparator. Methods Aspergillus isolates were screened for resistance using azole-agar. Aspergillus isolates that screened positive and all Candida isolates underwent EUCAST broth microdilution testing. Isolates were categorized as wild-type (wt) or non-wt, adopting EUCAST epidemiological cut-off values (ECOFFs) (where available) or wt upper limits (wtULs; two two-fold dilutions above the MIC₅₀). The CYP51A gene was sequenced for non-wt Aspergillus fumigatus isolates. Itraconazole and posaconazole MICs were determined for selected Aspergillus isolates with isavuconazole MIC ≥2 mg/L. Results Isavuconazole MIC₅₀ (range) (mg/L) against Candida species were: Candida albicans: ≤0.03 (≤0.03 to >4), Candida dubliniensis: ≤0.03 (≤0.03), Candida glabrata: ≤0.03 (≤0.03–4), Candida krusei: 0.06 (≤0.03–0.5), Candida parapsilosis: ≤0.03 (≤0.03–0.06), Candida tropicalis: ≤0.03 (≤0.03 to >4), Saccharomyces cerevisiae (anamorph: Candida robusta): ≤0.03 (≤0.03–0.5). Non-wt isavuconazole/voriconazole MICs were found for C. albicans: 0.8/1.0%, C. dubliniensis: 0/1.8%, C. glabrata: 14.9/9.5%, C. krusei: 2.7/1.4%, C. parapsilosis: 1.7/1.8%, C. tropicalis: 14.3/19.1% and S. cerevisiae: 10.0/0%. Isavuconazole MIC₅₀ (range) (mg/L) against Aspergillus species were: A. fumigatus: 1 (≤0.125 to >16), Aspergillus niger: 2 (1–8), Aspergillus terreus: 1 (0.25–8), Aspergillus flavus: 1 (0.5–2), Aspergillus nidulans: ≤0.125 (≤0.125–0.25). Non-wt isavuconazole/voriconazole MICs were found for 13.7/15.2% A. fumigatus, 4.9/0% A. niger and 48.2/22.2% A. terreus. Conclusion Isavuconazole displayed broad in vitro activity, similar to that of voriconazole. Up to 15% of C. glabrata, C. tropicalis and A. fumigatus isolates were non-wt, reflecting increased resistance at a reference centre and technical issues. Significant CYP51A alterations were reliably detected applying the isavuconazole breakpoint.