Microbial infections have become a global threat to drug-tolerant phenomena due to their biofilm formatting capacity. In many cases, conventional antimicrobial drugs fail to combat the infection, thus necessitating the discovery of some alternative medicine. Over several decades, plant metabolites have played a critical role in treating a broad spectrum of microbial infections due to its low cytotoxicity. Andrograpanin, a secondary metabolite, is a diterpenoid present in the leaf of Andrographis paniculata. In this study, andrograpanin (0.15 mM) exhibited significant inhibition on biofilm production by Pseudomonas aeruginosa in the presence of gentamicin (0.0084 mM). The impaired production of extracellular polymeric substances and several virulence factors of Pseudomonas aeruginosa were investigated to understand the mechanism of action mediated by andrograpanin. The structural alteration of biofilm was evaluated by using fluorescence microscopy, atomic force microscopy and field emission scanning electron microscopy. The in silico molecular simulation studies predicted interaction of andrograpanin with quorum sensing proteins such as RhlI, LasI, LasR, and swarming motility protein BswR of Pseudomonas aeruginosa. Overall the studies indicate that andrograpanin could be used as a therapeutic molecule against biofilm development by Pseudomonas aeruginosa.