The current treatment of leishmaniasis is confronted with significant challenges, including limited efficacy, adverse effects, and parasite resistance to drugs. The search for alternative therapeutic options, including the utilisation of natural products, has demonstrated considerable promise. In this study, the antileishmanial activity of the flavonoid hesperetin against Leishmania donovani, the causative agent of visceral leishmaniasis, was reported for the first time. Hesperetin was obtained through the hydrolysis of hesperidin and subsequently subjected to chemical characterisation via Infrared and NMR spectroscopy. The antileishmanial activity and cytotoxicity against RAW 264.7 macrophages were evaluated using the MTT colorimetric assay. In order to investigate the potential mechanisms of action, in vitro acetylcholinesterase inhibition assays and molecular docking analyses were conducted. Hesperetin showed an antipromastigote effect (IC