A diverse range of α-cyano bis(indolyl)chalcones (21a-r) were synthesized in high yields (90-95%) through the l-proline catalysed reaction of appropriate aldehydes and 3-cyanoacetylindoles. Formation of α-cyano bis(indolyl)chalcones involves readily available starting materials, catalytic l-proline, environmentally benign and metal-free conditions. The prepared eighteen α-cyano bis(indolyl)chalcones 21a-r were screened against prostate, breast, epithelial cancer cells and found to be non-cytotoxic to normal HEK293 cells. The α-cyano bis(indolyl)chalcones 21a (3.9 μM), 21c (7.5 μM), 21i (2.2 μM) and 21o (5.9 μM) displayed good cytotoxicity against C4-2 cells, whereas, derivatives 21c (1.23 μM), 21h (5.23 μM), and 21l (2.5 μM) showed selective cytotoxicity against 22Rv1 cells. With broad spectrum of activity (0.98-5.6 μM), the compound 21j was found to increase the endogenous level of ROS, upregulate the level of p-53 and c-jun besides mitochondrial dysfunction, cause apoptosis.