The pterocarpanquinone LQB-118, previously demonstrated to be effective in vivo via oral delivery, was investigated for its mechanism in selective parasite killing. Oxidative stress in Leishmania amazonensis was analysed by evaluating reactive oxygen species (ROS) production (2′,7′-dichlorodihydrofluorescein diacetate) and the loss of mitochondrial membrane potential (ΔΨ_m) using rhodamine, JC-1 and MitoCapture. Ultrastructural analysis was performed using transmission electron microscopy (TEM). DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). Treatment with LQB-118 induced ROS production in the promastigotes of L. amazonensis in a concentration-dependent manner for the first 4 h and was sustained for 24 h. TEM analysis revealed several alterations typical of apoptosis. Promastigotes presented a reduction of ΔΨ_m after 24 h of incubation with 2.5 μM (18.7%), 5 μM (63.7%) or 10 μM (70.7%) LQB-118. A sub-G₀/G₁ cell cycle phenotype was observed in 21%–83% of the promastigotes incubated with 1.25–10 μM LQB-118. Concentration-dependent DNA fragmentation was observed in promastigotes treated with 2.5–10 μM LQB-118, and selective DNA fragmentation was observed in intracellular amastigotes after 72 h with 2.5 μM treatment. Our results suggest that LQB-118 selectively induces ROS-triggered and mitochondria-dependent apoptosis in this parasite.