ASP2397 is a new compound with a novel and as-yet-unknown target different from that of licensed antifungal agents. It has activity against Aspergillus and Candida glabrata. We compared its in vitro activity against wild-type and azole-resistant A. fumigatus and A. terreus isolates with that of amphotericin B, itraconazole, posaconazole, and voriconazole. Thirty-four isolates, including 4 wild-type A. fumigatus isolates, 24 A. fumigatus isolates with alterations in CYP51A TR/L98H (5 isolates), M220 (9 isolates), G54 (9 isolates), and HapE (1 isolate), and A. terreus isolates (2 wild-type isolates and 1 isolate with an M217I CYP51A alteration), were analyzed. EUCAST E.Def 9.2 and CLSI M38-A2 MIC susceptibility testing was performed. ASP2397 MIC50 values (in milligrams per liter, with MIC ranges in parentheses) determined by EUCAST and CLSI were 0.5 (0.25 to 1) and 0.25 (0.06 to 0.25) against A. fumigatus CYP51A wild-type isolates and were similarly 0.5 (0.125 to >4) and 0.125 (0.06 to >4) against azole-resistant A. fumigatus isolates, respectively. These values were comparable to those for amphotericin B, which were 0.25 (0.125 to 0.5) and 0.25 (0.125 to 0.25) against wild-type isolates and 0.25 (0.125 to 1) and 0.25 (0.125 to 1) against isolates with azole resistance mechanisms, respectively. In contrast, MICs for the azole compounds were elevated and highest for itraconazole: >4 (1 to >4) and 4 (0.5 to >4) against isolates with azole resistance mechanisms compared to 0.125 (0.125 to 0.25) and 0.125 (0.06 to 0.25) against wild-type isolates, respectively. ASP2397 was active against A. terreus CYP51A wild-type isolates (MIC 0.5 to 1), whereas MICs of both azole and ASP2397 were elevated for the mutant isolate. ASP2397 displayed in vitro activity against A. fumigatus and A. terreus isolates which was independent of the presence or absence of azole target gene resistance mutations in A. fumigatus. The findings are promising at a time when azole-resistant A. fumigatus is emerging globally.