Twenty 2,3-disubstituted quinazolin-4(3H)-one derivatives 1e20 were successfully synthesized in moderate to good yields (25e82%). Their syntheses were based on a one pot tandem ring opening procedure followed by iodine-catalyzed oxidative cyclization of isatoic anhydride with aldehydes, using water as the only solvent under both classical and microwave irradiation conditions. Cytotoxicity assays of the prepared compounds against three human cancer cell lines (HeLa, MCF-7, and A549) indicated that 2, 3, and 20 possessed moderate activities against MCF-7 cells (IC50 ¼ 47.2 mM, 43.9 mM, and 44.9 mM, respectively). Good cytotoxic activities against A549 cells were observed for 3 and 8 with IC50 values of 30.7 mM and 29.8 mM, respectively, which were comparable to the positive control, 5-fluorouracil (5-FU, IC50 ¼ 27.9 mM). Furthermore, compound 4 exhibited slightly stronger activity (IC50 ¼ 23.6 mM) than the positive control 5-FU against the A549 cell line.