Monitoring antifungal susceptibility patterns for new or established antifungals is imperative. Antifungal resistance is frequent in molds, frequently leading to invasive mold infections (IMIs) in immunocompromised patients with high morbidity and mortality. Limited availability of effective antifungals for treatment of IMIs in China is an enormous challenge. The purpose of this study was to monitor in vitro antifungal resistance profiles of mold isolates from China, with a particular focus on evaluating in vitro isavuconazole (ISA) activity against these isolates, contributing to the treatment guidance in clinics. We evaluated the in vitro activity of ISA and its comparators (voriconazole VOR and amphotericin B AMB) against 131 clinical isolates of spp. ( = 105) and order ( = 26) collected between 2017 and 2020 from China. ISA and VOR exhibited similar in vitro activity against spp., with minimum inhibitory concentration (MIC) of 1 µg/mL and MIC of 2 µg/mL, respectively. Overall, AMB was less active than azoles against spp. (MIC : 4 µg/mL, MIC : 8 µg/mL). Against the order, ISA demonstrated MIC of 0.5 µg/mL and MIC of 1 µg/mL; however, one strain each of and were resistant to ISA (MICs: >8 µg/mL). VOR exhibited little or no activity (MIC : 8 µg/mL, MIC : >8 µg/mL) against the order, whereas AMB had MIC and MIC of 1 µg/mL. This was the first multicenter, in vitro study conducted in China and demonstrated the excellent activities of ISA against most species of the order. MIC indicated an advantage over currently available azole antifungals, positioning ISA as a potential alternative to VOR for clinical management of IMIs. As with other antimicrobials, clinicians should employ stewardship and best practices in relation to potential resistance to new azole antifungals.