A series of Michael-type analogues were generated on the C-ring of α-santonin (α-methylene-γ-butyrolactone) upon reaction with various thiols. All the thiol adducts synthesized were evaluated for their anticancer activity against four human cancer cell lines (PC-3, HCT-15, A-549 and MCF-7). Bioassay results indicated that even though most of the synthesized compounds exhibited a good anticancer activity against various cancer cells in vitro, some of the compounds like 9e, 9g and 9q were found to be the most promising analogues in this series, with compound 9e showing IC50 values of 1.5 μM, 0.6 μM, 2.4 μM and 1.2 μM on PC-3, MCF-7, A-549 and HCT-116 cell lines respectively. Further, flow cytometry studies showed that MCF-7 cells treated with the compounds 9e, 9g and 9q were arrested in the sub G1 phase of the cell cycle in a concentration dependent manner. These lead molecules were further studied for NF-κB, p65 transcription factor inhibitory activity which confirmed concentration dependent inhibition against NF-κB, p65 with analogue 9e showing 57% inhibition at 2 μM, 9g showing 62% inhibition at 3 μM and 9q showing 54% inhibition at 2 μM concentration.