Leishmaniasis is a neglected tropical disease that still infects thousands of people per year throughout the world. The occurrence of resistance against major treatments for this disease causes a healthcare burden in low-income countries. Eugenol is a phenylpropanoid that has shown in vitro antileishmanial activity againstLeishmania mexicana mexicana(Lmm) promastigotes with an IC50of 2.72 µg/mL and a high selectivity index. Its specific mechanism of action has yet to be studied. We prepared large unilamellar vesicles (LUVs), mimickingLmmmembranes, and observed that eugenol induced an increase in membrane permeability and a decrease in membrane fluidity at concentrations much higher than IC50. The effect of eugenol was similar to the current therapeutic antibiotic, amphotericin B, although the latter was effective at lower concentrations than eugenol. However, unlike amphotericin B, eugenol also affected the permeability of LUVs without sterol. Its effect on the membrane fluidity ofLmmshowed that at high concentrations (≥22.5× IC50), eugenol increased membrane fluidity by 20–30%, while no effect was observed at lower concentrations. Furthermore, at concentrations below 10× IC50, a decrease in metabolic activity associated with the maintenance of membrane integrity revealed a leishmaniostatic effect after 24 h of incubation withLmmpromastigotes. While acidocalcisomes distribution and abundance revealed byTrypanosoma bruceivacuolar H+pyrophosphatase (TbVP1) immunolabeling was not modified by eugenol, a dose-dependent decrease of lipid droplets assessed by the Nile Red assay was observed. We hereby demonstrate that the antileishmanial activity of eugenol might not directly involve plasma membrane sterols such as ergosterol, but rather target the lipid storage ofLmm.