Biologically active quinoline-3,4-dihydropyrimidin-2(1H)-one hybrids as novel monastrol analogs, were designed via molecular hybridization and structural modification rational. The synthesis was easily accessible via classical Biginelli reaction in good yields. Investigation of in vitro cytotoxicity was achieved through MTT cell line assay. The results revealed that the target derivatives showed a spectrum of potential cytotoxic effects on HepG-2, HCT-116, MCF-7, and PC3 cell lines, taking 5-FU as standard antitumor agents. Compounds 7e (R = tert-butoxy) and 7f (R = 2-methylpropyloxy) showed the greatest cytotoxic efficacy among the tested hybrids on the investigated cell lines; in which 7e showed IC50 values range of (11.57 μM - 19.05 μM), while 7f showed IC50 values range (14.16 μM - 19.96 μM), in comparison with the standard 5-FU (IC50 from 5.3 μM to 8.3 μM). The introduced quinoline-3,4-dihydropyrimidin-2(1H)-one hybrids with their readily straightforward structural modification, may serve as a potential cytotoxic antitumor candidate after suitable optimization.