The currently used treatments for leishmaniasis, a neglected parasitic disease, are associated with several side effects, high cost and resistance of the Leishmania parasites. Here we evaluated in vitro and in vivo the antileishmanial activity of five antimalarial drugs against Leishmania amazonensis. Mefloquine was effective against promastigotes in axenic cultures and showed an IC50 (concentration giving half-maximal inhibition) value of 8.4±0.7 mM. In addition, mefloquine, chloroquine and hydroxychloroquine were active against intracellular amastigotes in macrophageinfected cultures, presenting IC50 values of 1.56±0.19 mM, 0.78±0.08 mM and 0.67±0.12 mM, respectively. The ultrastructural analysis of chloroquine- or mefloquine-treated amastigotes showed an accumulation of multivesicular bodies in the cytoplasm of the parasite, suggesting endocytic pathway impairment, in addition to the formation of myelin-like figures and enlargement of the Golgi cisternae. CBA mice were infected with L. amazonensis in the ear dermis, and treated by oral and/or topical routes with chloroquine and mefloquine. Treatment of L. amazonensisinfected mice with chloroquine by the oral route reduced lesion size, which was associated with a decrease in the number of parasites in the ear, as well as the parasite burden in the draining lymph nodes. In contrast, mefloquine administration by both routes decreased the lesion size in infected mice without causing a reduction in parasite burden. Our results revealed a promising antileishmanial effect of chloroquine and suggest its use in cutaneous leishmaniasis treatment.