Antibiotic resistance caused by Gram-positive bacteria is a growing global human health threat. Selective discrimination and eradication of Gram-positive bacteria and their biofilms is challenging. Therapeutic strategies with multiple modes of action are urgently needed to address the increase in Gram-positive bacteria-resistant nosocomial infections. In this work, we have presented rationally designed aggregation-induced emission (AIE)-active cationic cyclometalated iridium(III) complexes derived from 2-phenylquinoline and 2,2'-bipyridine ligands for Gram-positive antibacterial studies. The AIE properties of these complexes were exploited for selective discrimination between Gram-positive and Gram-negative bacteria. These complexes displayed good antimicrobial activity against critical Gram-positive ESKAPE pathogens with minimum inhibitory concentrations in the low micromolar range but were inactive against Gram-negative pathogens. Importantly, the complexes can inhibit biofilm formation and eradicate bacteria from mature biofilms, which are major causes of persistent infections and antibiotic resistance and are more difficult to eliminate. In addition, these complexes showed low hemolytic activity against mammalian cells and a high therapeutic index, indicating good selectivity. Interestingly, the complexes kill bacteria through a variety of modes of mechanism, including ROS generation, cell membrane disruption, and depolarization and the loss of bacterial membrane integrity. These findings offer opportunities for designing metal AIEgens to treat Gram-positive bacterial infections effectively.