Ten bis(alkylpyridinium)alkane compounds were tested for antifungal activity against 19 species (26 isolates) of yeasts and molds. We then determined the MICs and minimum fungicidal concentrations (MFCs) of four of the most active compounds (compounds 1, 4, 5, and 8) against 80 Candida and 20 cryptococcal isolates, in comparison with the MICs of amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, and caspofungin, using Clinical Laboratory and Standards Institutes broth microdulition M27-A3 (yeasts) or M38-A2 (filamentous fungi) susceptibility protocols. The compounds were more potent against Candida and Cryptococcus spp. (MIC range, 0.74 to 27.9 microg/ml) than molds (0.74 to 59.7 microg/ml). MICs against Exophiala were 0.37 to 5.9 microg/ml and as low as 1.48 microg/ml for Scedosporium but >or=25 microg/ml for zygomycetes, Aspergillus, and Fusarium spp. Compounds 1, 4, 5, and 8 exhibited good fungicidal activity against Candida and Cryptococcus, except for Candida parapsilosis (MICs of >44 mug/ml). Geometric mean (GM) MICs were similar to those of amphotericin B and lower than or comparable to fluconazole GM MICs but 10- to 100-fold greater than those for the other azoles. GM MICs against Candida glabrata were <1 microg/ml, significantly lower than fluconazole GM MICs (P<0.001) and similar to those of itraconazole, posaconazole, and voriconazole (GM MIC range of 0.4 to 1.23 microg/ml). The GM MIC of compound 4 against Candida guilliermondii was lower than that of fluconazole (1.69 microg/ml versus 7.48 microg/ml; P=0.012). MICs against Cryptococcus neoformans and Cryptococcus gattii were similar to those of fluconazole. The GM MIC of compound 4 was significantly higher for C. neoformans (3.83 mug/ml versus 1.81 microg/ml for C. gattii; P=0.015). This study has identified clinically relevant in vitro antifungal activities of novel bisalkypyridinium alkane compounds.