Antimicrobial resistance has posed a serious health concern worldwide, which is mainly due to the excessive use of antibiotics. In this study, gold nanoparticles synthesized from the plant Tinospora cordifolia were used against multidrug-resistant Pseudomonas aeruginosa. The active components involved in the reduction and stabilization of gold nanoparticles were revealed by gas chromatography-mass spectrophotometry(GC-MS) of the stem extract of Tinospora cordifolia. Gold nanoparticles (TG-AuNPs) were effective against P. aeruginosa at different concentrations (50,100, and 150 µg/mL). TG-AuNPs effectively reduced the pyocyanin level by 63.1% in PAO1 and by 68.7% in clinical isolates at 150 µg/mL; similarly, swarming and swimming motilities decreased by 53.1% and 53.8% for PAO1 and 66.6% and 52.8% in clinical isolates, respectively. Biofilm production was also reduced, and at a maximum concentration of 150 µg/mL of TG-AuNPs a 59.09% reduction inPAO1 and 64.7% reduction in clinical isolates were observed. Lower concentrations of TG-AuNPs (100 and 50 µg/mL) also reduced the pyocyanin, biofilm, swarming, and swimming. Phenotypically, the downregulation of exopolysaccharide secretion from P. aeruginosa due to TG-AuNPs was observed on Congo red agar plates.