The present study aims to assess the in vitro antileishmanial activity of Myrtle (Pistacia khinjuk) essential oil against Leishmania major promastigotes. The in vitro antileishmanial effects of Khinjuk essential oil against L. major promastigotes were performed using MTT method. The IC50 values were also calculated by Probit test in SPSS software. The obtained results showed that myrtle extract was significantly inhibited promastigote growth of L. major based on a dose and time dependent manner. The measured IC50 values for Khinjuk essential oil and MA as control drug against promastigote forms of L. major were 8.9 μl/mL and 44.6 μg/mL, respectively. The obtained results showed antileishmanial effects of Khinjuk against promastigote forms of L. major. However, further studies will be needed to confirm these results by checking in the animal models as well as volunteer human.