Eugenia unifloraL. is a member of the Myrtaceae family and is commonly known as Brazilian cherry tree. In this study, we evaluated the chemical composition ofEugenia unifloraL. essential oil (EuEO) by using gas chromatography-mass spectrometry (GC-MS) and assessed its anti-Leishmaniaactivity. We also explored the potential mechanisms of action and cytotoxicity of EuEO. Thirty-two compounds were identified, which constituted 92.65% of the total oil composition. The most abundant components were sesquiterpenes (91.92%), with curzerene (47.3%),γ-elemene (14.25%), andtrans-β-elemenone (10.4%) being the major constituents. The bioactivity shown by EuEO against promastigotes (IC₅₀, 3.04 μg·mL⁻¹) and amastigotes (IC₅₀, 1.92 μg·mL⁻¹) suggested significant anti-Leishmaniaactivity. In the cytotoxicity determination, EuEO was 20 times more toxic to amastigotes than to macrophages. Hemolytic activity was 63.22% at the highest concentration tested (400 μg·mL⁻¹); however, there appeared to be no toxicity at 50 μg·mL⁻¹. While the data show that EuEO activity is not mediated by nitric oxide production, they do suggest that macrophage activation may be involved in EuEO anti-Leishmaniaactivity, as evidenced by increases in both the phagocytic capacity and the lysosomal activity. More studies are needed to determinein vivoactivity as well as additional mechanisms of the anti-Leishmaniaactivity.