The cytotoxic effect of the aqueous extract from Verbena officinalis, was evaluated in vitro on DHD/K12/PROb rat colonic epithelial cell line and HCT-116 human colon adenocarcinoma cell line. In both cell lines, the IC50 values were lower than 20 mu g/mL after 72 h of treatment. Bioassay guided fractionation led to the isolation of 12 phenylethanoid glycosides with anti-proliferative activity, five of them are being reported for the first time. The new compounds were elucidated as 4 ''-acetyl-O-isoverbascoside, 2 '',4 ''-diacetyl-O-verbascoside, 3"',4"'-diacetyl-O-isoverbascoside, 4"',6 ''-diacetyl-O-betonyoside A and 3"',4"'-diacetyl-O-betonyoside A. The IC50 results suggest that antiproliferative activity is determined by not only the number of acetyl-groups but also their position in the aliphatic rings. Compounds exhibiting vicinal acetyl-groups in the sugar rings such as 3"',4"'-diacetyl-O-isoverbascoside and 3"',4"'-diacetyl-O-betonyoside A are particularly strong cytotoxic compounds against both cell lines. This investigation indicated that diacetyl-phenylethanoids might be valuable as cancer chemopreventive agents.