Synthesis of 1,8-naphthalimide derivatives and biochemical studies using them as anti-cancer and cellular imaging agents have attracted significant attention in recent years. This study aimed to synthesize two new fluorescent narrow rim-functionalized 4-sulfo-1,8-naphthalimide derivatives of calix[4]arenes (4 and 7) and examination of their cytotoxic properties for cancerous cells. For this purpose, p-tert-butylcalix[4]arene bearing dihydrazide (3) or diaminopropyl (6) units on its narrow rim were reacted with 4-sulfo-1,8-naphthalic anhydrate. The structure of these synthesized compounds has been characterized using 1H-NMR, 13C-NMR, and FTIR techniques. To investigate the anti-cancer properties, compounds 4, 7, and N,N-dimethylaminoethyl-1,8-naphthalimide (8) were tested on the human colorectal cancer cell line (DLD-1) as well as healthy colon epithelial cell line (CCD-18Co). The IC50 values of 4 and 7 were determined as 12.95 μM and 16.13 μM, respectively, on DLD-1 cells. Furthermore, following the treatment of CCD-18Co cells with 4 and 7, the IC50 values were determined as 508 μM and 269 μM, respectively. However, it was observed that compound 8 had no cytotoxic effect on both DLD-1 cells and CCD-18Co cells. Furthermore, the fluorescence imaging of human colorectal cancer cells treated with the synthesized compounds (4, 7, and 8) was performed on living cells.