Violacein is a natural pigment, a pyrrolidone, and a bisindole derived from the condensation of two tryptophan molecules, which gives a blue violet color to several gram-negative violacein-producing bacteria. Violacein production provides a competitive advantage against antagonistic species or predators. In addition, the compound has antibacterial, antifungal, antiviral, and antioxidant activities. Several studies on colon, breast, and head and neck cancer lines have already demonstrated the anti-proliferative potential of violacein. Bladder cancer is one of the most common types of cancer in urology. The therapeutic approach is mainly based on surgery, chemotherapy, and immunotherapy. The aim of the present study was to evaluate the anti-proliferative activity of violacein against the human bladder cancer cell lines, HTB4 (T24) and HTB9 (5637), using low-grade and high-grade transitional cell carcinoma models, respectively, which has never been assayed before. For this purpose, the potential violacein anti-proliferative effect on T24 and 5637 cells was evaluated by studying the cell viability, proliferation, cell cycle, and caspase-3 activation. The results showed that violacein had anti-proliferative activity in the two cell lines, which was greater for the second-stage bladder cancer cell line (5637), and a different mode of action against the two cell lines.