Inflammatory bowel disease (IBD) is a chronic condition of the intestine with unknown etiology involving multiple immune, genetic and environmental factors. We were interested to examine the effect of total extract from Zataria multiflora Boiss, a folk medicinal plant on prevention and treatment of experimental IBD. Z. multiflora was administered (400, 600, 900 p.p.m.) through drinking water to IBD mice induced by intrarectal administration of acetic acid. Prednisolone was used as the standard drug for comparison. Biochemical, macroscopic and microscopic examinations of colon were performed. Biochemical evaluation of inflamed colon was done using assay of myeloperoxidase (MPO) activity and thiobarbituric acid reactive substances (TBARS) concentration as indicators of free radical activity and cell lipid peroxidation. The activity of MPO and lipid peroxidation products (TBARS) increased in acetic acid-treated groups while recovered by pretreatment of animals with Z. multiflora (400–900 p.p.m.) and prednisolone. Z. multiflora (600 and 900 p.p.m.) and prednisolone-treated groups showed significantly lower score values of macroscopic and microscopic characters when compared with the acetic acid-treated group. The beneficial effect of Z. multiflora (900 p.p.m.) was comparable with that of prednisolone. The antioxidant, antimicrobial and anti-inflammatory potentials of Z. multiflora might be the mechanisms by which this herbal extract protects animals against experimentally induced IBD. Proper clinical investigation should be carried out to confirm the activity in human.