A new compound 6-formamide-chetomin (1) together with chetomin (2) was isolated from solid fermentation products of the endophytic fungus Chaetomium sp. M336, which were elucidated by extensive spectroscopic analyses, including 1D and 2D NMR, and HR-ESI-MS experiments. The bioassay result showed that compound 1 had strong antibacterial activity against Escherichia coli, Staphylococcus aureus, Salmonella typhimurium ATCC 6539 and Enterococcus faecalis with minimum inhibitory concentration (MIC) value of 0.78 mu g/mL; meanwhile it exhibited strong cytotoxicity with IC50 values of 21.627.1 nM against cell lines HeLa, SGC-7901 and A549.