Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic bacterium commonly found in wound infections and airways of cystic fibrosis patients. P. aeruginosa readily forms biofilms which can reduce the efficacy of antibiotics used to eradicate the pathogen. We have previously shown that a Specialized Pro-resolving Mediator (SPM), Lipoxin A4 (LxA) is a quorum sensing inhibitor which can reduce P. aeruginosa virulence. In this study, we examined the direct actions of LxA and RvD on P. aeruginosa biofilm formation and virulence gene expression. The influence of LxA on antibiotic efficacy and the combined effects on biofilm formation were also investigated. LxA and RvD reduced P. aeruginosa biofilm formation and virulence gene expression. LxA increased ciprofloxacin inhibition on biofilm formation but did not affect ciprofloxacin’s action on non-adherent bacteria. On the other hand, LxA increased bacterial killing action of imipenem but did not affect imipenem’s action on biofilm. We also found that LxA can increase ciprofloxacin’s bacterial killing ability in established biofilm. Together these results suggest that LxA has direct effects on P. aeruginosa biofilm formation and can increase antibiotic efficacy directly.Copyright © 2020. Published by Elsevier Inc.