Colon cancer is one of the most common cancer-related deaths. Drug resistance is one of the biggest challenges in cancer treatment. Numerous pharmacological and biochemical investigations have documented the benzimidazole ring's anticancer, anti-inflammatory, and antioxidant properties. Within the scope of our project, the effect of newly synthesized benzimidazolium salt (BS) on cell proliferation was tested with MTT assay, and its effect on apoptosis and cell cycle was tested with annexin V and PI in the two different colon cancer cell lines (HT-29 and DLD-1). Our study examined the expressions of some genes related to apoptosis and, additionally, caspase activities with the multicaspase kit. BS showed an antiproliferative effect at lower doses in HT-29 colon cancer cells. When HT-29 cells were exposed to a 20 µM dosage, they showed increased caspase activity and apoptosis compared to DLD-1 cells. HT-29 accumulated in the G2/M phase of the cell cycle, whereas DLD-1 cells accumulated more in the S phase. In HT-29 cells, colony formation was inhibited; however, in DLD-1 cells, this effect was insufficient. Based on the apoptosis-death pathway, BS is expected to have anti-cancer effects. As a result of this work, this chemical was thoroughly examined in two different colon cancer cell lines, and additional, more comprehensive initiatives are being planned in light of the information obtained from this study.