The aqueous distillate of Origanum onites L. (Labiatae) was reported to have various ethnomedical uses including effects on the gastrointestinal tract. The effects of three different doses (0.1, 0.2 and 0.4 mL) of the aqueous distillate of Origanum onites on isolated rat fundus, duodenum and ileum against acetylcholine induced contractions were investigated in this study. The aqueous distillate inhibited contractions in a dose dependent manner. Inhibitions were lowest on fundus. Carvacrol, being the major compound of the test substance, did not inhibit acetylcholine induced contractions of the isolated rat fundus, indicating the presence of other active principles including menthane diols. As being one of the first pharmacological studies on aqueous distillates, a possible pharmacological activity of cis-p-menth-4-ene-1,2-diol and 3,7-dimethyl-l-octen-3,7-diol is proposed in this study.