Two previously undescribed steroidal alkaloids, compounds 1–2, were isolated from the ripe fruits of Solanum nigrum, along with seven known metabolites (3–9). Based on spectroscopic and chemical evidence, including IR, NMR, and HR-ESI-MS analyses, the structures of the isolated compounds were elucidated as 12β-hydroxy-(3β,22α,25R)-spirosol-5-en-27-acid-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→3)]-β-D-galacopyranoside and 12β-hydroxy-(3β,22α,25R)-spirosol-5-en-27-acid-3-O-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranoside. Four steroidal alkaloids (compounds 1–2 and 4–5) were tested for their anti-proliferative effects against the HT-29, A549, and Lewis cell lines. Both of the previously isolated compounds inhibited the proliferation of these three cell lines in a dose-dependent manner, with the most significant effect being in the A549 cells, but neither reached IC50 at 50 μM. These results revealed that S. nigrum had weak cytotoxicity, indicating its clinical safety as a traditional anti-tumor herbal medicine. © 2022 Phytochemical Society of Europe