In nature, bacteria rarely exist as single, isolated entities, but rather as communities comprised of many other species including higher host organisms. To survive in these competitive environments, microorganisms have developed elaborate tactics such as the formation of biofilms and the production of antimicrobial toxins. Recently, it was discovered that the Gram-negative bacterium Pseudomonas aeruginosa, an opportunistic human pathogen, produces an antibiotic, 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione (C-12-TA), derived from one of its quorum sensing molecules. Here, we present a comprehensive study of the expanded spectrum Of C-12-TA antibacterial activity against microbial competitors encountered by P. aeruginosa in nature as well as significant human pathogens. The mechanism of action Of C-12-TA was also elucidated, and C-12-TA was found to dissipate both the membrane potential and the pH gradient of Gram-positive bacteria, correlating well with cell death. Notably, in stark contrast to its parent molecule 3-oxo-dodecanoyl homoserine lactone (3-oxo-C-12-HSL), neither activation of cellular stress pathways nor cytotoxicity was observed in human cells treated with C-12-TA. Our results suggest that the QS machinery of P. aeruginosa has evolved for a dual-function, both to signal others of the same species and also to defend against host immunity and competing bacteria. Because of the broad-spectrum antibacterial activity, established mode of action, lack of rapid resistance development, and tolerance by human cells, the C-12-TA scaffold may also serve as a new lead compound for the development of antimicrobial therapeutics.