Abstract: Background and Objective: Hydatid cyst is the larval stage of the tapeworm Echinococcus granulosus which causes one of the most important zoonotic helminthic diseases worldwide. Using scolicidal agents before surgery may prevent recurrence of infection, however many scolicidal agents may cause serious side effects. Previously it has been proven that methanolic extract of Zataria multiflora has scolicidal effect in vitro and in vivo. However, the exact mechanism by which Z. multiflora exerts its scolicidal effect is unknown. Methodology: Therefore, the destructive effect of total methanolic extract of Z. multifora on the germinal layer of hydatid cyst was investigated. The germinal layer together with laminated layer of hydatid cyst were collected from livers of sheep and were exposed to 10, 20 and 30 mg mL–1 concentrations of the methanolic extract of Z. multiflora for 20, 30 and 60 min. Viability of the protoscolices was confirmed by 0.1% eosin staining. Results: This therapeutic regimen resulted in discontinuity, destruction and irregularity of the cells in the germinal layer, degeneration and destruction of protoscolices, detachment of the germinal layer from the laminated layer and vacuolation of the laminated layer. Conclusion: The effects of methanolic extract of Z. multiflora were mainly related to its concentration but variation in time of exposure had slight effect on its effect on germinal layer and protoscoleces.