The leishmanicidal activity of a series of 4‐aminoquinoline (AMQ) derivatives was assayed against Leishmania amazonensis. This activity against the intracellular parasite was found stronger than for L. amazonensis promastigotes. Neither compound was cytotoxic against macrophages. The compound AMQ‐j, which exhibited a strong activity against promastigotes and amastigotes of L. amazonensis (IC50 values of 5.9 and 2.4 μg/mL, respectively) and similar leishmanicidal activity to reference drugs, was chosen for studies regarding its possible mechanism of action toward parasite death. The results showed that the compound AMQ‐j induced depolarization of the mitochondrial membrane potential in promastigotes and in L. amazonensis‐infected macrophages, but not in uninfected macrophages. Furthermore, the depolarization of the mitochondrial membrane potential was dose dependent in infected macrophages. We have established that promastigotes and L. amazonensis‐infected macrophages treated with AMQ‐j were submitted to oxidative stress. This is in line with the increase in the level of reactive oxygen species (ROS). Leishmania amazonensis‐infected macrophages treated with AMQ‐j did not show a significant increase in the production of nitric oxide. Our results indicate the effective and selective action of AMQ‐j against L. amazonensis, and its mechanism of action appears to be mediated by mitochondrial dysfunction associated with ROS production.