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The application of nanotechnology in medicine is gaining popularity due to its ability to increase the bioavailability and biosorption of numerous drugs. Chrysin, a flavone constituent of <i>Orocylumineicum vent</i> is well-reported for its biological properties. However, its therapeutic potential has not been fully exploited due to its poor solubility and bioavailability. In the present study, chrysin was encapsulated into chitosan nanoparticles using TPP as a linker. The nanoparticles were characterized and investigated for their anti-biofilm activity against <i>Staphylococcus aureus</i>. At sub-Minimum Inhibitory Concentration, the nanoparticles exhibited enhanced anti-biofilm efficacy against <i>S. aureus</i> as compared to its bulk counterparts, chrysin and chitosan. The decrease in the cell surface hydrophobicity and exopolysaccharide production indicated the inhibitory effect of the nanoparticles on the initial stages of biofilm development. The growth curve analysis revealed that at a sub-MIC, the nanoparticles did not exert a bactericidal effect against <i>S. aureus</i>. The findings indicated the anti-biofilm activity of the chrysin-loaded chitosan nanoparticles and their potential application in combating infections associated with <i>S. aureus</i>.