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This study was aimed at investigating the leishmanicidal and immunomodulatory of oregano essential oil (OEO) from Origanum vulgare on experimental Leishmania amazonensis-model through in silico, in vitro, and in vivo approaches. For this purpose, drug-likeness prediction of OEO main components in silico was assessed, the leishmanicidal activity against promastigotes and amastigote-infected macrophages, as well its microbicide and immunomodulatory mechanisms in vitro, and the in vivo treatment on L. amazonensis-infected mice. It was demonstrated that OEO containing carvacrol, thymol, γ-terpinene, p-cymene, and β-caryophyllene as the main components is a good candidate for oral and topical drug development according to the in silico study, acting against L. amazonensis in vitro and in vivo in murine model. OEO acted on promastigote forms, triggering a combination of autophagic, apoptotic, and …