Current antifungal agents cover a majority of opportunistic fungal pathogens; however, breakthrough invasive fungal infections continue to occur and increasingly involve relatively uncommon yeasts and molds, which often exhibit decreased susceptibility. APX001A (manogepix) is a first-in-class small-molecule inhibitor of the conserved fungal Gwt1 protein. This enzyme is required for acylation of inositol during glycosylphosphatidylinositol anchor biosynthesis. APX001A is active against the major fungal pathogens, i.e., (except ), , and hard-to-treat molds, including and In this study, we tested APX001A and comparators against 1,706 contemporary clinical fungal isolates collected in 2017 from 68 medical centers in North America (37.3%), Europe (43.4%), the Asia-Pacific region (12.7%), or Latin America (6.6%). Among the isolates tested, 78.5% were spp., 3.9% were non- yeasts, including 30 (1.8%) var. isolates, 14.7% were spp., and 2.9% were other molds. All isolates were tested by CLSI reference broth microdilution. APX001A (MIC , 0.008 μg/ml; MIC , 0.06 μg/ml) was the most active agent tested against sp. isolates; corresponding anidulafungin, micafungin, and fluconazole MIC values were 16- to 64-fold higher. Similarly, APX001A (MIC , 0.25 μg/ml; MIC , 0.5 μg/ml) was ≥8-fold more active than anidulafungin, micafungin, and fluconazole against var. Against spp., AXP001A (50% minimal effective concentration MEC , 0.015 μg/ml; MEC , 0.03 μg/ml) was comparable in activity to anidulafungin and micafungin. isolates (>98%) exhibited a wild-type phenotype for the mold-active triazoles (itraconazole, posaconazole, and voriconazole). APX001A was highly active against uncommon species of , non- yeasts, and rare molds, including 11 isolates of spp. (MEC values, 0.015 to 0.06 μg/ml). APX001A demonstrated potent activity against recent fungal isolates, including echinocandin- and fluconazole-resistant strains. The extended spectrum of APX001A was also notable for its potency against many less common but antifungal-resistant strains. Further studies are in progress to evaluate the clinical utility of the methyl phosphate prodrug, APX001, in difficult-to-treat resistant fungal infections.