Withanolides are a diverse class of natural products with notable biological activities, including anticancer properties. This study expands knowledge about the genus Athenaea as a source of bioactive compounds against cancer, focusing on four species (A. fasciculata, A. martiana, A. tomentosa, and A. velutina).Dichloromethane and ethanol extracts from these species were evaluated for cytotoxicity against breast cancer (MCF-7) and melanoma (MV3) cell lines, as well as non-tumor cell lines (HEK-293 and BGM), using the MTT assay. The most cytotoxic extract was fractionated, and withanolide composition was analyzed by HPLC-MS/MS. Additionally, the antiproliferative activity of isolated withanolides—namely withalutin, withacnistin, and withacnistin acetate—was assessed in MV3 cells via Ki-67 immunofluorescence assay.Cytotoxic activity was observed for the ethanol extract of A. fasciculata and both extracts of A. velutina. The ethyl acetate fraction demonstrated superior cytotoxicity compared to other fractions, with withanolides, including glycosylated derivatives, identified in all fractions. Isolated withanolides exhibited significant antiproliferative and cytotoxic effects in tumor models.This is the first report describing the cytotoxicity of A. fasciculata in tumor models and the antiproliferative effects of three molecules isolated from A. velutina. These findings emphasize the potential of withanolides as anticancer agents and highlight the value of Brazilian biodiversity. This study lays the groundwork for preclinical investigations into developing new melanoma treatments.