A series of 2-arylquinazolinones with structural homology to known 3-arylisoquinolines were designed and synthesized in order to develop safe, effective, and selective cytotoxic agents targeting topoisomerases (topos). 2-Arylquinzolinones with various substitutions on the aromatic rings were obtained by thermal cyclodehydration/dehydrogenation on reacting anthranilamides and benzaldehydes. The compounds had superior topo I-inhibitory activities but were generally inactive against topo IIα. Among the 6-methyl-, 6-amino-, and 7-methylquinazolinones, 6-amino-substituted derivatives displayed potent cytotoxicity at submicromolar to nanomolar concentrations against human colorectal adenocarcinoma cells (HCT-15), human ductal breast epithelial tumor cells (T47D), and cervical cancer cells (HeLa). There was a good correlation between topo I inhibition and the cytotoxic effects of 6-aminoquinazolinones. Docking models demonstrated that topo I inhibition by these compounds is owing to intercalation and H-bond interactions with the DNA bases and amino acid residues at the enzymatic site.