Breast cancer affects millions of women annually worldwide and is the leading cause of cancer death in women. Various bioactive phytochemicals based on natural products are considered to be an important source of chemopreventive agents. In this study we report-to our knowledge for the first time-9 phytochemicals isolated by nuclear magnetic resonance spectroscopy and mass spectrometry from the acetic ether extract of Pleurotus nebrodensis and identified as (1) ergosterol, (2) uracil, (3) ergosterol-3-O-β-D-glucopyranoside, (4) cerevisterol, (5) cerebroside B, (6) 5'-methylthioadenosine, (7) adenosine, (8) hypoxanthine, and (9) uridine. Their bioactivities were screened with an MTT assay using breast cancer MCF-7 cells in vitro. As a result, about half of the isolated compounds demonstrated moderate or strong inhibitory activity in a concentration-dependent manner. Among them, compound 1 (ergosterol) exhibited superior activity and the lowest half-maximal inhibitory concentration (112.65 μmol/L). Further mechanistic study elucidated that compound 1 led to significant S-phase cell cycle arrest and induced apoptosis in MCF-7 cells. Our study shows the mycochemical composition of the P. nebrodensis mushroom and provides guidance for use of compound 1 as a promising lead in cancer therapy.