Cutaneous leishmaniosis is a major worldwide public health problem with annual incidence of 1.5 million cases across 98 countries. Treatment still relies on the use of chemical drugs with increasing resistance and side effects. The aim of this study was to investigate the anti-leishmanial effect of the hydroalcoholic Thymbra spicata extract on Leishmania major (L. major) promastigotes. In this study, 1x105 L. major promastigotes were cultured in 96-well plates and treated with different concentrations of hydroalcoholic T. spicata extract (12.5 to 400 mug/ml) then incubated at 25 degrees C for 24, 48 and 72 hours. Lethal percentage of promastigotes in each well was determined. RPMI 1640 medium containing L. major promastigotes with glucantime or without any treatment were used as positive and negative controls respectively. The 50 lethal concentration (LC50) of T. spicata extract and glucantime was calculated by GraphPad Prism software. The results indicated a significant decrease in the number of promastigotes treated with T. spicata extract and glucantime in comparison with negative control (P<0.0001). LC50 values for T. spicata extract were 18.49, 8.58, and 1.64 mug/ml after 24, 48 and 72 hours, respectively. In addition, anti-leishmanial effect of T. spicata extract and glucantime were dependent on concentration (P<0.0001). Our study revealed T. spicata extract as an herbal product against L. major promastigotes. However, more investigations are needed to find its antileishmanial activity in vivo and clinical trial studies