Quorum sensing (QS) is a cell-to-cell communication that is used by bacteria to regulate collective behaviors. Quorum sensing controls virulence factor production in many bacterial species and it is regarded as an attractive target to combat bacterial pathogenicity, especially against antibiotic-resistant bacteria. Chlorogenic acid (CA), abundant in fruits, vegetables, and Chinese herbs, processes multiple activities. In this research, we explored its quorum sensing quenching activity. In Pseudomonas aeruginosa, CA significantly inhibited the formation of biofilm, the ability of swarming, and virulence factors including protease and elastase activities and rhamnolipid and pyocyanin production. CA showed similar inhibitory effects in Chromobacterium violaceum on its biofilm formation, swarming motility, chitinolytic activity and violacein production. We examined the expression of QS-related genes inP.aeruginosa and found these genes were all downregulated by CA treatment. Computational modeling revealed that CA can form hydrogen bonds with all three QS receptors. Caenorhabditis elegans and mouse infection models were employed to explore the anti-virulence ability of CA and its effect on pathogenesis process in vivo. CA extended the survival period and reduced the quantity of P. aeruginosa in nematode gut, showing a moderate protective effect on C. elegans. In mice wound model, CA-treated groups showed an accelerating healing rate and the bacteria number in wound area was also decreased by CA treatment. It is suggested by our research that CA has potential to be used as an anti-virulence factor in P. aeruginosa infection.