The high prevalence of extended-spectrum β-lactamases (76.3 %) and metallo-β-lactamases (7.3 %) amongst the bacteria Pseudomonas aeruginosa is a critical problem that has set forth an enormous therapeutic challenge. The suggested role of nanoparticles as next generation antibiotics, and inadequate information on antibacterial activity of aluminium oxide nanoparticles has led us to investigate the green synthesis of aluminium oxide nanoparticles (Al2O3 NPs) using leaf extracts of lemongrass and its antibacterial activity against extended-spectrum β-lactamases and metallo-β-lactamases clinical isolates of P. aeruginosa. The synthesized Al2O3-NPs were characterized by scanning electron microcopy, high resolution-transmission electron microscopy, atomic force microscopy, X-ray diffraction, Zeta potential, and differential light scattering techniques. The X-ray diffraction data revealed the average size of the spherical Al2O3-NPs as 34.5 nm. The hydrodynamic size in Milli Q water and Zeta potential were determined to be 254 nm and +52.2 mV, respectively. The minimal inhibitory concentration of Al2O3-NPs was found to be in the range of 1,600-3,200 µg/ml. Treatment at concentrations >2,000 µg/ml, resulted in complete growth inhibition of extended-spectrum β-lactamases and metallo-β-lactamases isolates. Scanning electron microcopy analysis revealed the clusters of nanoparticles attached to the bacterial cell surface, causing structural deformities in treated cells. High resolution-transmission electron microscopy analysis confirmed that nanoparticles crossed the cell membrane to become intracellular. The interaction of nanoparticles with the cell membrane eventually triggered the loss of membrane integrity, most likely due to intracellular oxidative stress. The data explicitly suggested that the synthesized Al2O3-NPs can be exploited as an effective bactericidal agent against extended-spectrum β-lactamases, non-extended-spectrum β-lactamases and metallo-β-lactamases strains of P. aeruginosa, regardless of their drug resistance patterns and mechanisms. The results elucidated the clinical significance of Al2O3-NPs in developing an effective antibacterial therapeutic regimen against the multi-drug resistant bacterial infections. The use of leaf extract of lemongrass for the synthesis of Al2O3-NPs appears to be cost effective, nontoxic, eco-friendly and its strong antibacterial activity against multi-drug resistant strains of P. aeruginosa offers compatibility for pharmaceutical and other biomedical applications.