Pseudomonas aeruginosa is one of the most significant opportunistic infections in clinical setting and acquires resistance to diverse antibiotics. Honokiol (HON) is a natural bioactive compound isolated from the bark and seed cones of the Magnolia tree and acquires several benefits to health. The purpose of this research is to assess the antibacterial activity and anti-virulence properties of HON against P. aeruginosa. The effect of HON on the bacterial cell membrane as well as on bacterial virulence was estimated in vitro and in vivo. A docking investigation was carried out to determine the HON’s affinity to quorum sensing (QS) receptors and the influence of HON on QS expression’s genes. The outcome of the combination of HON and different antibiotics was performed. The resistance induction test was done to evaluate the HON's probability of inducing resistance. The effect of HON on mailman RBCs (red blood cells) was done to estimate the HON's safety. The results showed that HON damaged the bacterial cell membrane and reduced PMF and ATP synthesis. HON exhibited significant in vitro anti-virulence and anti-biofilm effects at sub-MIC and protected mice in vivo. These actions were attributed to its downregulation of QS-encoding genes and interfering with QS receptors. HON showed minimal impact on RBCs, indicating safety, and low likelihood of resistance induction. When mixed with antibiotics, HON revealed synergistic effectiveness against P. aeruginosa. In summary, HON shows potential as an effective adjuvant in treating severe P. aeruginosa infections.