Piper amalago L. leaves were extracted with supercritical carbon dioxide and compressed propane under different conditions, and with chloroform by the conventional maceration method. These methods were compared for the pyrrolidine alkaloid content. Supercritical carbon dioxide (SFE-CO₂) at 313 K and 12.55 MPa showed the highest selectivity for the main compound (600.53 mg/g of extract). A gradient high-performance liquid chromatography (HPLC) method was developed and validated to quantify the alkaloid N-[7-(3',4'-methylenedioxyphenyl)-2(Z),4(Z)-heptadienoyl]pyrrolidine in the extracts. The HPLC method showed linearity, precision and accuracy, allowing the quantitative analysis of the alkaloid in all the samples. All the extracts were tested against the promastigote and intracellular amastigote forms of Leishmania amazonensis. The antileishmanial activity was evaluated in terms of inhibitory concentration for 50% of protozoa (IC₅₀). The cytotoxicity was also evaluated against J774A1 macrophages, and the cytotoxic concentrations for 50% of macrophages were obtained (CC₅₀). The SFE-CO₂ (313 K; 12.55 MPa) extract showed the highest antileishmanial activity with the following IC₅₀ values of 16 and 7 µg/mL against the promastigotes and intracellular amastigotes forms, respectively. The extract showed low cytotoxicity with a CC₅₀ value of 93 µg/mL.