Honokiol, a bioactive compound isolated from the cone and bark of Magnolia officinalis, has been shown to have various activities including inhibition of the growth of Candida albicans. We investigated the roles of the Hsp90-calcineurin pathway in the antifungal activity of honokiol. The pharmacologic tool was employed to evaluate the effects of Hsp90 and calcineurin in the antifungal activity of honokiol. We also evaluated the protective effects of the calcineurin inhibitor cyclosporin A (CsA) on honokiol-induced mitochondrial dysfunction by the fluorescence staining method. The Hsp90 inhibitor potentiated the antifungal activity of honokiol. A C. albicans strain with the calcineurin gene deleted displayed enhanced sensitivity to honokiol. However, co-treatment with calcineurin inhibitor CsA attenuated the cytotoxic activity of honokiol due to the protective effect on mitochondria. Our results provide insight into the action mechanism of honokiol.