Background: Pseudomonas aeruginosa (P. aeruginosa) is the source of serious nosocomial infections, the most common of which is ventilator-associated pneumonia. P. aeruginosa infections continue to pose a substantial therapeutic problem. The expression of several virulence genes and the formation of biofilms in bacteria are caused by quorum sensing, a density-dependent cell-to-cell communication mechanism. Anti-biofilm chemicals prevent the synthesis of the polymer matrix, limit cell adhesion and attachment, reduce the generation of virulence factors, and obstruct the quorum sensing system. The present study focused on the antibiofilm activity of chrysin-fabricated silver nanoparticles (nano-chrysin) against P. aeruginosa. Materials and Methods: In our study, chrysin, which is a polyphenol, was fabricated with silver nanoparticles to make nano-chrysin. P. aeruginosa PAO1 strain was treated at sub-MIC concentrations with chrysin (50, 25, 12.5, and 6.25 μg mL-1), silver nanoparticles (6.26, 3.13, 1.56, and 0.78 μg mL-1) and formulated nano-chrysin (3.13, 1.56, 0.78, and 0.39μg mL-1) to find out the effectiveness of these compounds against biofilm formation. Results: Biofilm produced by P. aeruginosa PAO1 was found to be inhibited at sub-MIC concentrations (3.13, 1.56, 0.78, and 0.39 μg mL-1) of nano-chrysin having MIC value ranging between 50-3.13 μg mL-1 which is more potent than alone chrysin and silver nanoparticles. Conclusion: The data confirmed that nano-chrysin is effective in inhibiting biofilm formation, produced by P. aeruginosa.