Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative opportunistic pathogen posing serious risks to immunocompromised individuals due to its virulence factors and biofilm formation. This study evaluated the efficacy of methanol extract of Glycyrrhiza glabra (G. glabra) in mitigating P. aeruginosa PAO1 pathogenesis through in-vitro assays, including Minimum Inhibitory Concentration (MIC), biofilm assay, growth curve analysis, pyocyanin quantification, and molecular docking. The extract inhibited PAO1 growth at 5 mg/mL and demonstrated significant antibiofilm activity at sub-MIC levels, reducing biofilm formation by 50.22 %, 22.13 %, and 11.53 % at concentrations of 1.25 mg/mL, 0.625 mg/mL, and 0.312 mg/mL, respectively. Pyocyanin production was also significantly suppressed. Molecular docking revealed that 4-(4-Trifluoromethyl-benzoylamino)-benzoic acid and betulinic acid, identified in the extract, exhibited strong binding affinities (-6.4 kcal/mol and -6.9 kcal/mol) to the QS regulator 7XNJ. These findings underscore the potential of G. glabra as an antipathogenic agent against P. aeruginosa, warranting further investigation into its clinical applications.