The treatment of leishmaniasis relies primarily on highly toxic, parenterally administered drugs. Therefore, the search for more effective and safer drugs is considered a priority for leishmaniasis’ control. The antileishmanial activity of the oestrogen receptor modulator tamoxifen was previously described in experimental models of leishmaniasis. However, the mechanisms responsible for the antileishmanial activity of tamoxifen remain unknown. Since tamoxifen has been shown to affect plasma and intracellular membranes in tumour cells, the aim of this study was to investigate the activity of the drug on Leishmania amazonensis membranes. Through morphological analysis and labelling with propidium iodide and DiSBAC2(3), we demonstrated that tamoxifen led to plasma membrane depolarization without general membrane disruption or permeabilization. Tamoxifen also caused mitochondrial damage, with loss of membrane potential, as shown by Rhodamine 123 accumulation. Mitochondrial swelling followed, signalling the mitochondrial dysfunction. Therefore, the effect of tamoxifen on Leishmania is mediated, at least in part, by disorder in parasite's membranes. These alterations are sufficient to trigger a series of lethal events.