A series of novel 2-alkythio-4-chloro-N-[imino-(heteroaryl)methyl]benzenesulfonamide derivatives,8–24, were synthesized in the reaction of theN-(benzenesulfonyl)cyanamide potassium salts1–7with the appropriate mercaptoheterocycles. All the synthesized compounds were evaluated for their anticancer activity in HeLa, HCT-116 and MCF-7 cell lines. The most promising compounds,11–13, molecular hybrids containing benzenesulfonamide and imidazole moieties, selectively showed a high cytotoxic effect in HeLa cancer cells (IC50: 6–7 μM) and exhibited about three times less cytotoxicity against the non-tumor cell line HaCaT cells (IC50: 18–20 μM). It was found that the anti-proliferative effects of11,12and13were associated with their ability to induce apoptosis in HeLa cells. The compounds increased the early apoptotic population of cells, elevated the percentage of cells in the sub-G1 phase of the cell cycle and induced apoptosis through caspase activation in HeLa cells. For the most active compounds, susceptibility to undergo first-phase oxidation reactions in human liver microsomes was assessed. The results of the in vitro metabolic stability experiments indicated values of the factor t½for11–13in the range of 9.1–20.3 min and suggested the hypothetical oxidation of these compounds to sulfenic and subsequently sulfinic acids as metabolites.