LY303366 and MK-0991 (previously L-743,792) are new echinocandin derivatives with excellent broad-spectrum antifungal activity. We investigated the in vitro activity of LY303366, MK-0991, itraconazole, amnphotericin B, and 5-flucytosine against 51 clinical isolates of filamentous fungi, including Aspergillus flavus (10), A. fumigatus (12), Fusarium spp. (13), Rhizopus spp. (6), Pseudallescheria boydii (5), and one isolate each of Acremonium spp., A. niger, A terreus, Paecilomyces spp., and Trichoderma spp. In vitro susceptibility testing was performed using a microdilution broth method performed according to National Committee for Clinical Laboratory Standards guidelines. Ly303366 was two- to fourfold more active than MK-0991 against A. flavus, A. fumigatus, and Trichoderma spp. Both LY303366 and MK-0991 were considerably more active (MIC90 of 0.03-0.12 micrograms/mL) than itraconazole, amphotericin B, and 5-flucytosine against Aspergillus spp., but were less active than intraconazole and amphotericin B against Rhizopus spp. MK-0991 was more active than either LY303366 or intraconazole against Acremonium spp., Paecilomyces spp., and P. boydii. These data demonstrate promising activity of both LY303366 and MK-0991 against Aspergillus spp. and other species of filamentous fungi that are likely to be encountered clinically. Further in vitro and in vivo investigation is indicated.