© 2016 Elsevier B.V. and International Society of ChemotherapyAntifungal susceptibilities of non-Aspergillus filamentous fungal pathogens cannot always be inferred from their identification. Here we determined, using the Sensititre® YeastOne® YO10 panel, the in vitro activities of nine antifungal agents against 52 clinical isolates of emergent non-Aspergillus moulds representing 17 fungal groups in Australia. Isolates comprised Mucorales (n?=?14), Scedosporium/Lomentospora spp. (n?=?18) and a range of hyaline hyphomycetes (n?=?9) and other dematiaceous fungi (n?=?11). Excluding Verruconis gallopava, echinocandins demonstrated poor activity (MICs generally >8?mg/L) against these moulds. Lomentospora prolificans (n?=?4) and Fusarium spp. (n?=?6) demonstrated raised MICs to all antifungal drugs tested, with the lowest being to voriconazole and amphotericin B (AmB), respectively (geometric mean MICs of 3.4?mg/L and 2.2?mg/L, respectively). All Scedosporium apiospermum complex isolates (n?=?14) were inhibited by voriconazole concentrations of =0.25?mg/L, followed by posaconazole and itraconazole at =1?mg/L. Posaconazole and AmB were the most active agents against the Mucorales, with MIC90 values of 1?mg/L and 2?mg/L, respectively, for Rhizopus spp. For dematiaceous fungi, all isolates were inhibited by itraconazole and posaconazole concentrations of =0.5?mg/L (MIC90, 0.12?mg/L and 0.25?mg/L, respectively), but voriconazole and AmB also had in vitro activity (MIC90, 0.5?mg/L and 1?mg/L, respectively). Differences in antifungal susceptibility within species and between species within genera support the need for testing individual patient isolates to guide therapy. The Sensititre® YeastOne® offers a practical alternative to the reference methodology for susceptibility testing of moulds.