Natural products are some of the important sources of new anticancer drugs. The Brazilian flora is considered one of the most diverse in the word, although not many large-scale pharmacological and phytochemical studies have been conducted to date. With this in mind, in the present study we evaluated the antiproliferative activity of Solanum lycocarpum fruit glycoalkaloid extract (SL) and its major compounds, solamargine (SM) and solasonine (SS), against different tumor cell lines: murine melanoma (B16F10), human colon carcinoma (HT29), human breast adenocarcinoma (MCF-7), human cervical adenocarcinoma (HeLa), human hepatocellular liver carcinoma (HepG2) and human glioblastoma (MO59J, U343 and U251). The antiproliferative activity was evaluated using XTT assay and results were expressed as IC50. The most pronounced antiproliferative activity was observed for SM, with IC50 values ranging from 4.58 to 18.23 μg/mL. The lowest IC50 values were observed against HepG2, being 9.60 μg/mL for SL, 4.58 μg/mL for SM and 6.01 μg/mL for SS. Thus, SL, SM and SS demonstrated antiproliferative activity against the tumor cell lines tested, and were most effective against the HepG2 cell line.