Background: Calix[n]arenes have attracted great attention due to their biocompatibility and superior stability. When the necessary functional groups are attached to these compounds, they may have the potential to target tumor tissues. Benzimidazoles are among the anticancer drugs discovered in recent years. Aim: The aim of this study was to design and synthesise a series of calix[4]arenes-benzimidazoles. For comparison purposes, a benzimidazole derivative was synthesized by attaching it to the diester. The anticancer effects of these compounds were investigated by performing cell proliferation, apoptosis, and cell imaging studies on cancer cell lines. Methods: Some of the obtained compounds were synthesized by employing the methods available in literature studies, and the rest were synthesized by modifying previous methods. As a result, a total of 3 new fluorescent calix[4]arene-benzimidazole derivatives were synthesized. MTT was used for cell proliferation, and Annexin V was used for apoptosis studies. For confocal imaging studies, cells were treated with DAPI and MitoTracker dyes. Results: Four designed calix[4]arene-benzimidazoles were successfully synthesized and structurally confirmed by ¹H-NMR, ¹³C-NMR, and IR spectroscopy. The anticancer study on four synthesized compounds was performed. Bio-imaging studies were performed using confocal microscopy for the three successfully synthesized fluorescent compounds. Conclusion: CB5-a and CB5-c were found to be the most effective against MCF-7 cells and CB5-b against HT-29 cells in the MTT test. Apoptosis analyses also proved that these compounds inhibited the proliferation of cancer cells. As a comparison compound, the synthesized CB5-R proved to be less cytotoxic than the fluorescent compounds by the MTT method, and we found the cationic compound to bind to the calix[4]arene more effectively than the molecule’s binding to the diester.