A new sterol, ganolutol A (1), together with 4 known compounds 2 to 5, were isolated from the polypore fungus Ganoderma luteomarginatum collected in Myanmar. The chemical structures of the isolated compounds were determined based on extensive spectroscopic analyses in conjunction with comparisons with published data. Compounds 1 to 4 exhibited moderate cytotoxic activities against A549 (lung), MCF-7 (breast), and HeLa (cervical) human cancer cell lines, with IC50 values ranging from 10.1 to 86.6 μM.