Pseudomonas aeruginosa infections are versatile, persistent, and difficult to treat due to the ability to produce virulent factors, form biofilm, and resist antibiotics. This study has tested a novel protocol that targets Pseudomonas aeruginosa pathogenicity mechanisms and quorum sensing mechanisms to manage and control P. aeruginosa infections. The aim of this study is to assess the effectiveness of Azadirachta indica methanolic extracts in inhibiting P. aeruginosa biofilm, virulence factors, and quorum sensing. The findings showed that the A. indica extract has strong antibiofilm activity against P. aeruginosa at concentrations that are below what would normally stop the growth of the bacteria (1.56, 0.78, and 0.39 mg/mL). The results also showed that it has several antibiofilm effects that target swarming motility, aggregation, hydrophobicity, and the production of exopolysaccharide (EPS). Also, A. indica effectively stopped the production of several harmful substances, such as pyocyanin, rhamnolipids, and LasA protease. It's important to note that A. indica extract stops P. aeruginosa and Chromobacterium violaceum from making acyl-homoserine lactone and violacein, which shows that it can mess up quorum sensing mechanisms. The findings of this study suggest that A. indica extract is a promising choice for developing alternative therapeutic strategies aimed at controlling and treating biofilm-related infections in humans, animals, and plants.