A series of novel benzimidazole–triazole acetamide hybrids with different substitutions at the acetamide moiety were designed and synthesized in an effort to discover potential anticancer agents. The compounds were evaluated for their in vitro antiproliferative and cytotoxicity activities against two cancer cell lines (A549 and SW480) and a normal cell (MRC-5) using the MTT assay. The results revealed that most derivatives exhibited moderate to high levels of antiproliferative activity. Notably, derivative 9f emerged as the most potent antiproliferative agent with IC₅₀ values of 16.1 ± 1.1 and 19.7 ± 2.7 μM against A549 and SW480, respectively. Compound 9f showed significant selectivity towards A549 (SI = 7.5) and SW480 (SI = 6.1) cancer cells compared to the normal MRC-5. Furthermore, this compound exhibited much lower cytotoxicity than cisplatin and doxorubicin against the normal cells. The effects of 9f on cell cycle distribution and apoptosis induction in A549 cells were investigated using flow cytometry. The derivative significantly arrested cells in the S phase and remarkably induced apoptosis at the IC₅₀ concentration. Compound 9f was predicted to have suitable pharmacokinetics and low toxic effects as an anticancer candidate drug. The docking study demonstrated that compound 9f interacted with topoisomerase II-DNA, exhibiting a binding energy comparable to that of etoposide.