A chemical investigation of the ethyl acetate extract of fruit bodies of Vietnamese lingzhi, Ganoderma neo-japonicum, collected in Cat Tien National Park, Lam Dong province, has resulted in the isolation and structural elucidation of four fungal metabolites, ergosta-4,6,8(l4),22-tetraen-3-one (1), ganodermadiol (2), ergosterol (3) and ergosta-7,22-dien-3,beta-ol (4) through the use of various chromatographic and spectroscopic methods, including ESI-MS and 2D NMR. In addition, compounds 1, 3 and 4 showed moderate cytotoxic activity toward KB cell lines with their IC50 values of 12.68, 10.72 and 87.95 ug/mL, respectively.