Leishmaniasis is a vector-borne disease and one of the most significant neglected tropical diseases. Current anti-leishmanial treatments are often ineffective over extended periods and are associated with toxic side effects, highlighting the urgent need for new, effective, and safe alternative treatments for this infectious disease. The objective of this study was to evaluate the anti-leishmanial effects of a hydroalcoholic extract of Hypericum scabrum (H. scabrum), comparing its efficacy to that of the control drug glucantime against the standard strain of Leishmania major. The H. scabrum plants were collected from the western regions of Iran. A hydroalcoholic extract was prepared from the flower and stem of the plant using a maceration method. High-performance liquid chromatography analysis was conducted to identify the chemical compounds present in the extract. Promastigotes of L. major were cultured, and the anti-leishmanial activity of the extracts was assessed at concentrations ranging from 12.5 to 800 µg/ml using the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay. The half-maximal inhibitory concentration (IC50) values for the H. scabrum plant extract at 24, 48, and 72 h were 245.47, 141.25 and 85.11 μg/ml, respectively. The IC50 values for glucantime (the control drug) at 24 h, 48 h, and 72 h were 30.19, 21.37, and 12.58 μg/ml, respectively. While the H. scabrum extract exhibited a lower effect compared to the control drug, it still demonstrated a significant inhibitory effect on the promastigote form of L. major. Given that the plant extract of H. scabrum has demonstrated promising anti-leishmanial effects against L. major promastigotes, further studies are warranted to evaluate the efficacy of these extracts in animal models of leishmaniasis.