The aim of this study was to evaluate the phytochemicals, antioxidant, antibacterial and anti-quorum sensing (QS) properties of methanol extract of Muntingia calabura L. leaves against biofilm-forming strain Pseudomonas aeruginosa. The antioxidant potential of M. calabura methanol leaf extract was evaluated using a radical scavenging assay. Since the findings were so promising, the study aims to screen the phytochemical profiles using qualitative and quantitative approaches and to expand the investigation to anti-QS activity. The antibacterial activity was assessed by determining the minimum inhibitory concentration against pathogenic Gram-negative and Gram-positive biofilm-forming bacterial strains using an agar well diffusion plate method. Muntingia calabura methanol leaf extract was most potent against P. aeruginosa. The QS controlled virulence factors in P. aeruginosa is significantly inhibited by M. calabura leaf extract. The microscopy images revealed a major reduction in P. aeruginosa biofilm formation. Interestingly, M. calabura leaf extract was not toxic to Vero cell line. These findings make M. calabura a strong candidate for new antimicrobial and anti-QS herbal agents.