Chitosan is a natural polymer derived from chitin, a structural component of fungi, insects and shrimp, which exerts antimicrobial effects against bacteria and fungi. The aim of this study was to investigate the in vitro antifungal activity of low molecular weight chitosan (LMWC), and the potential synergy between chitosan and a currently used antifungal drug, fluconazole. The in vitro minimal inhibitory concentrations (MICs) of chitosan and fluconazole against 105 clinical Candida isolates were measured by the broth microdilution method. LMWC exhibited a significant antifungal activity, inhibiting over 89.9% of the clinical isolates examined (68.6% of which was completely inhibited). The species included several fluconazole-resistant strains and less susceptible species such as C. glabrata, which was inhibited at a concentration of 4.8 mg/l LMWC. Although some strains were susceptible at pH 7.0, a greater antifungal activity of LMWC was observed at pH 4.0. There was no evidence of a synergistic effect of the combination of LMWC and fluconazole at pH 7.0. This is the first report in which the antifungal activity of LMWC was investigated with clinical Candida strains. The use of LMWC as an antifungal compound opens new therapeutic perspectives, as the low toxicity of LMWC in humans supports its use in new applications in an environment of pH 4.0-4.5, such as a topical agent for vulvovaginal candidiasis.