Acinetobacter baumannii is a major human pathogen associated with multidrug-resistant nosocomial infections; its virulence is attributed to quorum-sensing-mediated biofilm formation, and disruption of biofilm formation is an attractive antivirulence strategy. Here, we report the first successful demonstration of biofilm disruption in a clinical isolate of A. baumannii S1, using a quorum-quenching lactonase obtained by directed evolution; this engineered lactonase significantly reduced the biomass of A. baumannii-associated biofilms, demonstrating the utility of this antivirulence strategy.