<i>Candida</i> spp. are common causative agents of mucocutaneous infections. New therapeutic antifungal drugs are needed to treat chronic disease as these are frequently clinically resistant to azols. Chitosan, among other possible vehicles for active compounds, shows an added value as it appears to have intrinsic antimicrobial properties. The aim of the present study was to evaluate the anti-<i>Candida</i> activity of a medium-molecular-weight chitosan hydrogel (CH), to clarify its possible mechanism of action and to evaluate its cytotoxicity on human fibroblasts. CH antifungal activity was assessed according to CLSI reference M27-A3 protocol; its mechanism of action was investigated by flow cytometry, and its cytotoxicity was studied by MTT assay. CH demonstrated a full inhibition of <i>C. tropicalis</i>, <i>C. krusei</i>, <i>C. guilliermondii</i> and <i>C. parapsilosis</i> growth while impairing <i>C. albicans</i> and <i>C. glabrata</i> viability. Flow cytometry tests showed that CH acts by inducing primary lesion of the cytoplasmic membrane. However, CH showed no cytotoxic effect upon human fibroblasts cells. Resistant strains will require new therapeutic approaches. Chitosan being a good carrier and having itself anti-<i>Candida</i> activity seems to be a promising vehicle to be used for the treatment of mucocutaneous candidosis.