Background: Solanum nigrum L. (SN), commonly known as black nightshade or Makoi in India, is well documented in Ayurveda, the indigenous system of medicine, for its medicinal properties. However, it has still not garnered considerable attention for modern therapeutic use. In the present study, in vitro activities of methanolic extract(s) of dried roots, stems and leaves of S. nigrum were evaluated against human cancer cell lines MDA, Hep G2, A549 and normal cell line Vero. Methods: The percentage viability of the cell lines was determined using MTT assay. Antibacterial activity of the extracts was tested against S. aureus and E. coli using disc diffusion method. Molecular docking studies were carried out to assess the inhibitory action of selected phytoconstituents belonging to the class of steroidal glycoalkaloids (SGAs) and steroidal saponins (SSs) against cytoskeletal proteins viz. actin (G- and F-), tubulin (alpha and beta) and vimentin. Results: HepG2 cells were found to be most susceptible to SN extracts. Leaf extract of SN showed significant anticancer activity against Hep G2 and MDA cells (IC50 values approx. 20 µg/ml). AGE of isolated DNA from treated cancer cells revealed characteristic ladder like fragmentation, a hallmark of apoptosis. HPLC profiling demonstrated the presence of alpha-solanine in all extracts. Molecular docking analysis revealed that SGAs displayed potent binding to the intermediate filament protein vimentin (Ki 1.0-8.1 µM) whereas the SSs displayed moderate to low binding to vimentin. Conclusion: SN methanolic extracts inhibited proliferation and induced apoptosis in human cancer cell lines and thus, warrant further investigation. [J Complement Med Res 2017; 6(4.000): 391-400]