INTRODUCTION: Leishmaniasis denotes a significant health challenge worldwide with no ultimate treatment. The current study investigated the biological effects of gamma-terpinene (GT) on Leishmania major in putative antileishmanial action, cytotoxicity, apoptosis induction, gene expression alteration, antioxidant activity, hemolysis, and ROS generation. METHODS: GT and meglumine antimoniate (MA) were probed alone and in combination (GT/MA) for their anti-leishmanial potentials using the MTT biochemical colorimetric assay and a model macrophage cell. In addition, their immunomodulatory properties were assessed by analyzing their effect on the transcription of cytokines related to Th1 and Th2 responses. GT and MA, alone and in combination, were also assessed for their potential to alter metacaspase gene expression in L. major promastigotes by real-time RT-PCR. The hemolytic potential of GT and MA-treated promastigotes were also measured by routine UV absorbance reading. Electrophoresis on agarose gel was employed to analyze genomic DNA fragmentation. RESULTS: GT demonstrated notable dose-dependent antileishmanial effects towards promastigotes and amastigotes of L. major. The IC values for GT against L. major promastigotes and amastigotes were 46.76 mM and 25.89 mM, respectively. GT was considerably safer towards murine macrophages than L. major amastigotes with an SI value of 3.17. Transcriptional expression of iNOS, JAK-1, Interleukin (IL-10), and TGF-β was meaningfully decreased, while the levels of metacaspase mRNA were increased. Results also confirmed GT antioxidant activities. Also, increased levels of intracellular ROS were observed upon treatment of promastigotes with GT. The gel electrophoresis result indicated slight DNA fragmentation in the treated promastigotes by both drugs. A weak hemolytic effect was also observed for GT. CONCLUSION: The results demonstrated that GT showed potent activity against L. major stages. It seems that its mechanism of action involves representing an immunomodulatory role towards upregulation of iNOS and JAK-1, while downregulation of IL-10 and TGF- β. Moreover, GT has an antioxidative potential and exerts its action through activating macrophages to kill the organism. Further in vivo and clinical studies are essential to explore its effect in future programs.