From the fruiting body of ectomycorrhizal fungi Cortinarius xiphidipus, sterols were identified from the crude extract and the cytotoxic effect of ergosta-4, 6, 8(14), 22-tetraen-3-one (ergone) was evaluated. Ten sterols including ergosta-3,5,7,9(11),22-pentaene, (22E)-ergosta-5,7,9(11),22-tetraen-3b-ol, (3β,22E)-ergosta-5,7,22-trien-3-ol, (22E)-ergosta-7,22-dien-3-ol, neoergosterol, (3β)-ergosta-5,8-dien-3-ol, (3β)-ergosta-7-en-3-ol, stigmasterol, stigmasterol 22,23-dihydro and (22E)-ergosta-4,6,8(14),22-tetraen-3-one were identified from the crude extract. The cytotoxic activity of the sterol fraction containing ergosta-4, 6, 8(14), 22-tetraen-3-one was assessed on four tumour cell lines (Neuro-2a, Saos-2, MCF7 and LNCaP-C42). The cytotoxic activity against the four tumour cell lines tested, being Neuro-2a and Saos-2 the most sensitive, with a half-maximal inhibitory concentration (IC) of 20.8 ± 2.2 and 27.8 ± 1.0 μg/mL, respectively. This is the first report of this Antarctic fungi collected in the Magallanes and Chilean Antarctica Region. This work represents a potential source for the development of anticancer drugs.