4-Hydroxycoumarins constitute the structural nucleus of many natural products, drugs, and pesticides. Promising biological properties in new families of synthetic coumarins were recently reported. Therefore, efficient synthesis of new benzopyrano[3,2-c]chromene-6,8-dione was undertaken and the structures of 15 compounds were confirmed by their IR, Mass, 1 H-NMR, and C, H, N analysis. Then, the cytotoxic activities of these compounds were assessed on four different human cancer cell lines (Raji, HeLa, LS180, and MCF-7). The results showed that these compounds had weak-to-moderate antitumoral activities and their IC50 ranged from 49 to more than 100 lM. Among the compounds 9,10-dihydro-7-(3 methoxyphenyl)-7H,11H-benzopyrano[3,2-c]chromene-6,8 dione [4k] demonstrated the highest activity. Furthermore, conformational analysis revealed that ortho substituents were clearly different from meta and para substituents.