A series of benzimidazolium salts having 2-cyanobenzyl (2-6), 2-(4-nitrophenyl) ethyl (7-10), (1,3-dioxoisoindolin-2-yl) butyl (11-17) or N-methylphthalimide (18-22) substitution were synthesized and characterized by spectroscopic and analytical techniques. The in vitro cytotoxic activity of these salts was tested against human colon cancer (DLD-1), human breast cancer (MDA-MB-231), and normal human noncancerous embryonic kidney (HEK-293T) cell lines using the MTT assay method. The viability rates of the cells were determined by confocal microscopy. The majority of the newly synthesized compounds, with the exception of compounds 2, 3, 8, 11, 12, and 17, displayed high cytotoxic activity against cancerous cells. In fact, compounds 6 and 7 exhibited better results than the positive control drugs, cisplatin and busulfan, against the MDA-MB-231 cells. Therefore, compounds 6 and 7 could be considered as anticancer drug candidates for further development.