The aim of this study was to individuate, by bioassay-guided fractionation, promising antifungal fractions and/or constituents from Hypericum perforatum subsp. angustifolium in vitro roots. Treatments with chitosan, O-carboxymethylchitosan (CMC) and its derivatives were used to improve xanthone production in the roots. The bioassay-guided fractionation of CMC-treated roots led to the individuation of an ethyl acetate fraction, containing the highest amount of xanthones (6.8%) and showing the best antifungal activity with minimal inhibitory concentration (MIC) values of 53.82, 14.18, and 36.52 mu g/ml, against Candida spp., Cryptococcus neoformans and dermatophytes, respectively. From this fraction the prenylated xanthone, biyouxanthone D has been isolated and represented the 44.59% of all xanthones detected. For the first time in the present paper biyouxanthone D has been found in H. perforatum roots and tested against C neoformans, dermatophytes, and Candida species. The xanthone showed the greatest antifungal activity against C neoformans and dermatophytes, with MIC values of 20.16, 22.63 mu g/ml. In conclusion, the results obtained in the present study demonstrated that CMC-treated Hpa in vitro root extracts represent a tool for the obtainment of promising candidates for further pharmacological and clinical studies. (c) 2013 Elsevier Masson SAS. All rights reserved.