Cancer treatment remains a significant challenge, with chemotherapy still being one of the most common therapeutic approaches. Based on our initial studies of symmetric monomethine cyanine dyes, which showed potential against colorectal cancer, this study explored several asymmetric cyanines, aiming to develop more potent and selective antitumor agents, particularly against colorectal cancer. In pursuit of this goal, we have designed, synthesized, and structurally characterized twelve new cyanine dyes. Their antiproliferative effects were then investigated in vitro against both tumor and non-tumor cell lines. Notably, the two most promising dyes in terms of potency and selectivity against Caco-2 colorectal cancer cells were derived from the combination of -methylbenzoxazole and -methylquinoline (dye ), as well as -ethylbenzothiazole and -ethyl-6-nitrobenzothiazole (dye ). The potential mechanisms behind their antiproliferative action were also explored, revealing that both dyes penetrate cells and localize within the cytoplasm and nucleus. Furthermore, dye was found to slightly induce apoptosis without causing significant cell cycle arrest, in contrast to dye , which increased the number of cells in the G0/G1 phase. Interestingly, both dyes exhibited marked topoisomerase II inhibitory effects, particularly cyanine , which may further explain their antiproliferative activity. Additionally, drug-likeness properties were predicted for both dyes. Overall, cyanine emerged as the most promising candidate for further investigation as a potential treatment for colorectal cancer.