Separation and enrichment of phenolics from peony flowers were performed to improve the anti-biofilm and antibacterial activities for the first time. Through several times of separation, the purity of phenolics components increased significantly, and the anti-biofilm and antibacterial activities of phenolics components against E. coli and S. aureus were also significantly improved. Finally, the phenolics of peony flowers in the eluent of silica gel column chromatography (PPF-ESGCC) were found to exhibit the highest anti-biofilm and antibacterial activities. The inhibition rates of PPF-ESGCC on biofilms of E. coli and S. aureus were 77.93%, and 87.03% respectively, at a very low concentration (1/2 MIC, 0.235 mg/mL). It was found that the biofilm inhibition was achieved by inhibiting their swimming, swarming, twitching motilities, exopolysaccharide (EPS) production, and quorum sensing (QS). Moreover, there was a positive dose-dependent relationship (r = 0.75 to 1) between the inhibition rates and concentrations of PPF-ESGCC during the critical biofilm-formation stage (1-3 days). Chemical composition analysis showed the PPF-ESGCC comprised of gallic acid, kaempferol-7-O-glucoside, and apigenin-7-O-glucoside. In conclusion, PPF-ESGCC exhibited strong inhibitory effect on biofilm formation and gallic acid, kaempferol-7-O-glucoside, and apigenin-7-O-glucoside might play a crucial role in inhibiting biofilm formation. Meanwhile, this study indicated that PPF-ESGCC, a new natural QS inhibitor and biofilm inhibitor, could be used as a novel intervention strategy to enhance the safety and quality of food.