Leishmaniasis remains a significant global health challenge, with limited therapeutic options and rising drug resistance. The repurposing of Food and Drug Administration approved antidepressants as amitriptyline, a widely used tricyclic drug, could offer a promising strategy for developing novel antileishmanial agents. This study investigates the in vitro activity of amitriptyline against promastigotes and intracellular amastigotes and explores its ultrastructural effects and potential in combination therapy. Amitriptyline was effective against the clinically relevant form, the intracellular amastigotes of , resulting in an EC value of 22 μM. Ultrastructural analyses revealed mitochondrial swelling following amitriptyline treatment, suggesting mitochondria as a key target. This structural damage, in the absence of observable plasma membrane disruption, supports the hypothesis that amitriptyline may specifically target intracellular organelles rather than initiating cell lysis through membrane destabilization. Additionally, amitriptyline induces mitochondrial membrane depolarization in , disrupting parasite energy homeostasis. Combination assays with standard drugs amphotericin B and miltefosine demonstrated additive interactions, reinforcing the potential of amitriptyline as complementary therapy. This work highlights the antileishmanial activity of tricyclic compounds and underscores the potential for further repositioning studies, broadening the assessment of clinically used, structurally related compounds.