Spongistatin 1 is a macrocyclic lactone polyether from the marine sponge Hyrtios erecta. The aim of this study was to evaluate the in vitro and in vivo antifungal efficacies and mechanism of spongistatin 1. Spongistatin 1 was fungicidal for the majority of 74 reference strains and clinical isolates, including those resistant to flucytosine, ketoconazole or fluconazole, and retained activity in the presence of human serum or at lowered pH. The duration of the postantifungal effect following 1 h exposure to one, four and eight times the minimal inhibitory concentration was strain-dependent. Spongistatin 1 was significantly more efficacious than amphotericin B in reducing kidney infectious burden in a murine model of disseminated candidiasis, and reduced the lung burden in a murine model of pulmonary cryptococcosis. When cryptococcal microtubules were visualized by fluorescence microscopy and iterative deconvolution, spongistatin 1 was shown to disrupt first cytoplasmic and then spindle microtubules in a time- and concentration-dependent manner. Microtubule disruption was accompanied by an abnormal distribution of nuclei in budding cells and an inhibition of cell division, resulting in cells arrested in a large-budded stage. Spongistatin 1 should be pursued as a potential antifungal agent and as a probe to study, major cellular processes.