ABSTRACTDue to the prevalence of biofilm‐related infections, which are mediated by bacterial quorum sensing, there is a critical need for materials and coatings that resist biofilm formation. We have developed novel anti‐biofilm coatings that disrupt quorum sensing in surface‐associated bacteria via the immobilization of acylase in polyurethane films. Specifically, acylase from Aspergillus melleus was covalently immobilized in biomedical grade polyurethane coatings via multipoint covalent immobilization. Coatings containing acylase were enzymatically active and catalyzed the hydrolysis of the quorum sensing (QS) molecules N‐butyryl‐L‐homoserine lactone (C4‐LHL), N‐hexanoyl‐L‐homoserine lactone (C6‐LHL), and N‐(3‐oxododecanoyl)‐L‐homoserine lactone (3‐oxo‐C12‐LHL). In biofilm inhibition assays, immobilization of acylase led to an approximately 60% reduction in biofilm formation by Pseudomonas aeruginosa ATCC 10145 and PAO1. Inhibition of biofilm formation was consistent with a reduction in the secretion of pyocyanin, indicating the disruption of quorum sensing as the mechanism of the coating activity. Scanning electron microscopy further showed that acylase‐containing coatings contained far fewer bacterial cells than control coatings that lacked acylase. Moreover, acylase‐containing coatings retained 90% activity when stored dry at 37°C for 7 days and were more stable than the free enzyme in physiological conditions, including artificial urine. Ultimately, such coatings hold considerable promise for the clinical management of catheter‐related infections as well as the prevention of infections in orthopedic applications (i.e., on hip and knee prostheses) and on contact lenses. Biotechnol. Bioeng. 2016;113: 2535–2543. © 2016 Wiley Periodicals, Inc.