The bioassay-guided fractionation of MeOH extract from Naematoloma fasciculare afforded a petroleum ether fraction (NFPF) and four known compounds, which showed good antitumor activities to inhibit MCF-7 cell line proliferation in vitro and tumor growth in $H_{22}$ implanted mice in vivo. In addition, a number of unsaturated aliphatic acids were identified in NFPF by GC analysis. These results showed that NFPF inhibits tumor growth through the activity of unsaturated aliphatic acids together with two active compounds, ergosterol peroxide (1: 62.17 mg/g in NFPF) and ergosterol (2: 3.13 mg/g in NFPF), and indicate the potential utility of NFPF as an antitumor drug.