Leishmaniasis is an infection caused by a protozoan parasite of the genus Leishmania and is the second most prevalent parasitic protozoal disease after malaria in the world. We report the in vitro leishmanicidal activity on promastigote forms of Leishmania amazonensis and cytotoxicity, using LLCMK2 cells, of the glycoalkaloids from the fruits of Solanum lycocarpum, determined by colorimetric methods. The alkaloidic extract was obtained by acid-base extraction; solamargine and solasonine were isolated by silica-gel chromatography, followed by reversed-phase HPLC final purification. The alkaloidic extract, solamargine, solasonine, as well as the equimolar mixture of the glycoalkaloids solamargine and solasonine displayed leishmanicidal activity against promastigote forms of L. amazonensis, whereas the aglycone solasodine was inactive. After 24 and 72 h of incubation, most of the samples showed lower cytotoxicities (IC50 6.5 to 124 μM) as compared to leishmanicidal activity (IC50 1.1 to 23.6 μM). The equimolar mixture solamargine/solasonine was the most active with an IC50 value of 1.1 μM, after 72 h. Likewise, solamargine was the most active after 24 h with an IC50 value of 14.4 μM, both in comparison with the positive control amphotericin B.