Leishmaniasis is an important neglected disease caused by protozoa of the genus Leishmania that affects more than 12 million people worldwide. Leishmaniasis treatment requires the administration of toxic and poorly tolerated drugs, and parasite resistance greatly reduces the efficacy of conventional medications. Apigenin (1), a naturally occurring plant flavone, has a wide range of reported biological effects. In this study, antileishmanial activity of 1 in vitro was investigated, and its mechanism of action against Leishmania amazonensis promastigotes was described. Treatment with 1 for 24 h resulted in concentration-dependent inhibition of cellular proliferation (IC50 = 23.7 μM) and increased reactive oxygen species (ROS) generation. Glutathione and N-acetyl-L-cysteine protected L. amazonensis from the effects of 1 and reduced ROS levels after the treatment. By contrast, oxidized glutathione did not reduce the levels of ROS caused by 1 by not preventing the proliferation inhibition. Apigenin 1 also induced an extensive swelling in parasite mitochondria, leading to an alteration of the mitochondrial membrane potential, rupture of the trans-Golgi network, and cytoplasmic vacuolization. These results demonstrate the leishmanicidal effect of 1 and suggest the involvement of ROS leading to mitochondrial collapse as part of the mechanism of action.