This paper reports the effects of BnSP-7 toxin, a catalytically inactive phospholipase A2 from Bothrops pauloensis snake venom, on Leishmania (Leishmania) amazonensis. BnSP-7 presented activity against promastigote parasite forms both in the MTT assay, with IC50 of 58·7 μg mL−1 of toxin, and a growth curve, inhibiting parasite proliferation 60–70% at concentrations of 50–200 μg mL−1 of toxin 96 h after treatment. Also, the toxin presented effects on amastigotes, reducing parasite viability by 50% at 28·1 μg mL−1 and delaying the amastigote–promastigote differentiation process. Ultrastructural studies showed that BnSP-7 caused severe morphological changes in promastigotes such as mitochondrial swelling, nuclear alteration, vacuolization, acidocalcisomes, multiflagellar aspects and a blebbing effect in the plasma membrane. Finally, BnSP-7 interfered with the infective capacity of promastigotes in murine peritoneal macrophages, causing statistically significant infectivity-index reductions (P < 0·05) of 20–35%. These data suggest that the BnSP-7 toxin is an important tool for the discovery of new parasite targets that can be exploited to develop new drugs for treating leishmaniasis.