A series of novel 1,4-naphthoquinones possessing pyrido[2,3-d]pyrimidine scaffolds were synthesized in very good yields using one-pot condensation of 2-hydroxy-1,4-naphthoquinone, aldehydes, and 2-substituted 4,6-diaminopyrimidine. The in vitro anti-proliferative activity of these novel compounds was evaluated in SGC7901 and HepG2 cell lines. Almost all the tested compounds showed manifested potent inhibitory activity against the two tested cancer cell lines.