Calf diarrhea is one of the most important diseases of dairy farms and causes significant economic damage to farmers. Antibiotic-resistant bacterial infection has become a serious concern as antibiotic use has increased, leading to increased use of medicinal herbs. This study was performed to evaluate in vitro and in vivo antimicrobial effect of Zataria multiflora on bovine Escherichia coli. Sixty calves with Escherichia coli diarrhea were selected through microbial culture results. In the first experiment, the antibacterial activities of hydro-alcoholic extract of different levels of Zataria multiflora were compared with sulfadimidine to screening against fecal coli using a standard protocol of Disc Diffusion Method (DDM). In the second experiment, the calves were randomly treated with hydro-alcoholic extract of Zataria multiflora or sulfadimidine. Clinical measurements including rectal temperature, fecal scores, and appetite were assessed. The results of this investigation revealed that a 20 μl of hydro-alcoholic extract of Zataria multiflora had antibacterial activity comparable to sulfadimidine. Our clinical results showed that hydro-alcoholic extract of Zataria multiflora had superior effects on rectal temperature and stool consistency than sulfadimidine. Also, the use of hydroalcoholic extract of this plant could improve the process of these parameters in calves. The findings show that a hydro-alcoholic extract of Zataria multiflora can be utilized to treat diarrhea caused by E. coli.