The search for new therapeutic agents from natural sources has been a constant for the treatment of diseases such as leishmaniasis. Herein, in vitro and in vivo pharmacological activities of pure major phenolic constituents (caffeic acid, chlorogenic acid, ferulic acid, quercetin, and rosmarinic acid) from Pluchea carolinensis against Leishmania amazonensis are presented. Pure compounds showed inhibitory activity against promastigotes (IC50 = 0.2-0.9 μg/mL) and intracellular amastigotes (IC50 = 1.3-2.9 μg/mL). Four of them were selected after testing against macrophages of BALB/c mice: caffeic acid, ferulic acid, quercetin, and rosmarinic acid, with selective indices of 11, 17, 10, and 20, respectively. Ferulic acid, rosmarinic acid, and caffeic acid controlled lesion size development and parasite burden in footpads from BALB/c experimentally infected mice, after five injections of compounds by intralesional route at 30 mg/kg every 4 days. Pure compounds from P. carolinensis demonstrated antileishmanial properties.