Microbial spoilage in nutrient-rich strawberries has led to significant food waste and economic loss in the food industry. The quorum-quenching enzyme AiiA is believed to disrupt communication between cells by inactivating AHLs-based quorum sensing signals. Chitosan, a biopolymer derived from chitin, is widely used as a coating film to inhibit bacterial growth and prolong food shelf life. The present study aims to investigate the individual and combined effects of AiiA and chitosan on the preservation of strawberries. AiiA was synthesized in vitro and the reaction conditions for the degradation of AHLs signals were optimized at temperatures ranging from 20 to 60 °C and a pH of 8.0. The present study provides evidence that AiiA effectively inhibited the processes of biofilm development, production of exopolysaccharides, and extracellular protease activity in Enterobacter sp. and Pseudomonas aeruginosa (P. aeruginosa). Utilizing the bioprotective characteristics of AiiA, we implemented it in the preservation of strawberries. Our experiments show that AiiA, when used alone, improved the brightness, redness, and hardness of strawberries infected by Enterobacter sp. When combined with chitosan, AiiA had a notably beneficial effect on the sensory quality, color, hardness, and soluble solids content of strawberries that were infected with Enterobacter sp. and P. aeruginosa. Both AiiA alone and AiiA combined with chitosan treatment effectively reduced bacterial and fungal counts in strawberries infected by P. aeruginosa and inhibited bacterial growth in those strawberries infected by Enterobacter sp. Our study provides evidence that AiiA, either alone or in combination with chitosan shows potential application in preserving agricultural products.