Pseudomonas aeruginosa, is one the major causative agents for nosocomial and biofilm mediated infections. The present study is an attempt to disarm the biofilm forming potential of PAO1 using gold nanoparticles bio-synthesized from Hemidesmus indicus L. root extract (HiAuNPs). The HiAuNPs were optimally synthesised using 1 mM HAuCl4 at 70 degrees C for 20 min with a maximum surface plasmon resonance (SPR) at 531 nm. The average size distribution, elemental composition, crystalline nature and the capping of functional biomolecules of HiAuNPs were ascertained by DLS, SEM-EDAX, XRD, FTIR and HPLC analyses respectively. HRTEM identified that HiA-uNPs were predominantly hexagonal with an average particle size of 37 nm. The thermal properties of HiAuNPs were studied using thermal gravimetric analysis. The minimum biofilm inhibitory concentration (300 mu g/mL) of HiAuNPs greatly mitigated the biofilm-forming ability as well as the virulence factors such as EPS, hydropho-bicity index, pyocyanin, swarming motility, surfactant production, catalase and haemolytic activity of PAO1, without affecting its growth. Moreover, HiAuNPs exhibited significant synergistic antibacterial activity with antibiotics. The cytotoxicity assessment unveiled the non-cytotoxic nature of HiAuNPs against the human em-bryonic kidney cell lines. Thus, the present study reports HiAuNPs as a nontoxic, potent antibiofilm agent against P. aeruginosa.