In this research, a series of novel diosgenin-1,4-quinone hybrids were synthesized and evaluated in antiproliferative assays against three human cancer cell lines (MCF-7, HepG2, and HeLa). Structure-activity relationship analysis revealed that the activities depended on the type of 1,4-quinone moiety. Among them, hybrid 11a exhibited significant cytotoxicity against the HepG2 cell line with a IC of 1.76 μM, which was 35-fold more potent than diosgenin (IC = 43.96 μM). Western blot analysis showed that hybrid 11a upregulated Bax, Cl-caspase-3/9, and Cl-PARP levels, and downregulated Bcl-2 level of HepG2 cell line. Meanwhile, hybrid 11a could increase the generation of intracellular reactive oxygen species. The molecular docking study revealed an interaction between hybrid 11a and NQO1 enzyme. Our present studies suggested that hybrid 11a as a potential substrate for NQO1 enzyme could be a promising anticancer agent for further investigation.