In this study, some benzimidazole-oxadiazole derivatives were synthesised and tested for theirin vitroanticancer activities on five cancer cell lines, including HeLa, MCF7, A549, HepG2 and C6. Their structures were elucidated by IR,H-1-NMR,C-13-NMR, 2 D-NMR and HRMS spectroscopic methods. Among all screened compounds;5a,5b,5d,5e,5k,5l,5nand5oexhibited potent selective cytotoxic activities against various tested cancer cell lines. Especially, compounds5land5nexhibited the most antiproliferative activity than Hoechst 33342 and doxorubicin against HeLa cell line, with IC(50)of 0.224 +/- 0.011 mu M and 0.205 +/- 0.010 mu M, respectively. Furthermore, these potent lead cytotoxic agents were evaluated in terms of their inhibition potency against Topoisomerase I and it was determined that selected compounds inhibited the Topoisomerase I. Docking studies were performed and probable interactions in the DNA-Topo I enzyme complex was determined.