Aims The aim of this study is to investigate the antibacterial activity of aluminium oxide nanoparticles (Al2O3 NPs) against multidrug-resistant clinical isolates of Escherichia coli and their interaction with cell envelope biomolecules. Methods and Results Al2O3 NPs were characterized by scanning electron microscope (SEM), high-resolution transmission electron microscope (HR-TEM) and X-ray diffraction (XRD) analyses. Antibacterial activity and interaction of Al2O3 NPs with E.coli and its surface biomolecules were assessed by spectrophotometry, SEM, HR-TEM and attenuated total reflectance/Fourier transform infrared (ATR-FTIR). Of the 80 isolates tested, about 64 (80%) were found to be extended spectrum beta-lactamase (ESBL) positive and 16 (20%) were non-ESBL producers. Al2O3 NPs at 1000 mu gml(-1) significantly inhibited the bacterial growth. SEM and HR-TEM analyses revealed the attachment of NPs to the surface of cell membrane and also their presence inside the cells due to formation of irregular-shaped pits and perforation on the surfaces of bacterial cells. The intracellular Al2O3 NPs might have interacted with cellular biomolecules and caused adverse effects eventually triggering the cell death. ATR-FTIR studies suggested the interaction of lipopolysaccharide (LPS) and L-alpha-Phosphatidyl-ethanolamine (PE) with Al2O3 NPs. Infrared (IR) spectral changes revealed that the LPS could bind to Al2O3 NPs through hydrogen binding and ligand exchange. The Al2O3 NPs-induced structural changes in phospholipids may lead to the loss of amphiphilic properties, destruction of the membrane and cell leaking. Conclusions The penetration and accumulation of NPs inside the bacterial cell cause pit formation, perforation and disorganization and thus drastically disturb its proper function. The cell surface biomolecular changes revealed by ATR-FTIR spectra provide a better understanding of the cytotoxicity of Al2O3 NPs. Significance and Impact of the Study Al2O3 NPs may serve as broad-spectrum bactericidal agents to control the emergent pathogens regardless of their drug-resistance mechanisms.