Background: Benzimidazole derivatives bearing pyridyl/pyrimidinyl piperazine moiety has attracted attention in medicinal chemistry and modern drug discovery since it exhibited a variety of biological activities, including anticancer activity. Objective: In this study, we designed and synthesized novel 1-[2-oxo-2-(4-substituted phenyl)ethyl]benzimidazol-2-yl)methyl 4-(2-pyridyl/pyrimidin-2-yl)piperazine-1-carbodithioate derivatives (2a-m). We also investigated their anticancer activities against A549 lung adenocarcinoma and C6 rat glioma cell lines. We further studied the selectivity of the compounds against the NIH/3T3 mouse embryonic fibroblast cell line. Cholinesterase inhibition effects of these compounds were also investigated to measure the relationship between anticancer activity and cholinesterases. Methods: The cytotoxic activities of these acquired thirteen final compounds were screened using MTT assay on A549, C6, and NIH/3T3 cell lines. Cell proliferation ELISA, BRDU (colorimetric) assay was used to measure the proliferation in replicative cells in which DNA synthesis occurs. Flow cytometric analysis was used to measure apoptotic cell percentages, caspase 3 activity, and mitochondrial membrane depolarised cell percentages. Results: Compounds 2e, 2f, and 2k were shown to be the most active antitumor agents with selective cytotoxicities (the results for A549 were 76.58 +/- 6.43, 55.13 +/- 5.75, and 32.94 +/- 3.02 mu M, respectively; and for C6 they were 86.48 +/- 3.60, 97.12 +/- 30.21, and 59.29 +/- 3.95 mu M, respectively), high DNA synthesis inhibition rates and high apoptotic cell percentages on both cell lines. Conclusion: The results showed that compounds 2e, 2f, and 2k have potential anticancer activity against A549 and C6 cell lines.