A series of 6-diethylaminoquinazolin-4(3H)-ones with bulky aryl rings at C2 were designed to cover the vacant space of ligand binding pocket of topoisomerase (topo) I-DNA complex. The desired derivatives were synthesized by thermal cyclodehydration/ dehydrogenation reactions of 5-diethylaminoanthranilamide with substituted aromatic aldehydes. The cytotoxicity of these compounds was evaluated against human epidermoid carcinoma (KB), hepatocellular carcinoma (Hep-G2), human lung carcinoma (LU-1) and human breast carcinoma cells (MCF-7). Most of the synthesized compounds exhibited more potent cytotoxicity than the standard anticancer agent, ellipticine. Among the tested compounds, quinazolinone 1l was the most cytotoxic against all cancer cell lines (IC50: 0.02–0.08 µM). Docking study showed that the new 2-aryl-6- diethylaminoquinazolinones possibly inhibit topo I activities to exhibit anticancer properties.