The present study reports the mechanism of the antileishmanial activity of quercetin against the intracellular amastigote form of Leishmania amazonensis. Treatment with 1 reduced the infection index in L. amazonensis-infected macrophages in a dose-dependent manner, with an IC₅₀ value of 3.4 μM and a selectivity index of 16.8, and additionally increased ROS generation also in a dose-dependent manner. Quercetin has been described as a pro-oxidant that induces the production of reactive oxygen species, which can cause cell death. Taken together, these results suggest that ROS production plays a role in the mechanism of action of 1 in the control of intracellular amastigotes of L. amazonensis.