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Bacterial biofilms are responsible for most clinical infections and show increased antimicrobial resistance. In this study, molecularly imprinted polymers (MIPs) were developed to specifically capture prototypical quorum sensing autoinducers [i.e., N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-CAHL)], interrupt quorum sensing, and subsequently inhibit biofilm formation of Pseudomonas aeruginosa, an important human nosocomial pathogen. The synthesis of MIPs was optimized by considering the amount and type of the functional monomers itaconic acid (IA) and 2-hydroxyethyl methacrylate (HEMA). IA-based MIPs showed high adsorption affinity toward 3-oxo-CAHL with an imprinting factor of 1.68. Compared to IA-based MIPs, the adsorption capacity of HEMA-based MIPs was improved fivefold. HEMA-based MIPs significantly reduced biofilm formation (by ∼65%), whereas biofilm suppression by IA-based MIPs was neutralized because of increased bacterial attachment. The developed MIPs represent promising alternative biofilm intervention agents that can be applied to surfaces relevant to clinical settings and food processing equipment.