To date very little studies are available in the literature on the interaction of Al2O3 nanoparticles with multidrug-resistant strains of Staphylococcus aureus. Considering the paucity of earlier reports the objective of present study was to investigate the antibacterial activity of Al2O3 NPs (<50 nm) against methicillin-resistant S. aureus and methicillin-resistant coagulase negative staphylococci by various methods. Al2O3 NPs were characterized by scanning electron microscopy, high-resolution transmission electron microscopy, and X-ray diffraction. The MIC was found to be in the range of 1,700–3,400 μg/ml. Almost no growth was observed at 2,000 μg/ml for up to 10 h. SEM micrograph revealed that the treated cells were significantly damaged, showed indentation on cell surface and clusters of NPs on bacterial cell wall. HR-TEM micrograph shows disruption and disorganization of cell membrane and cell wall. The cell membrane was extensively damaged and, most probably, the intracellular content has leaked out. Al2O3 NPs not only adhered at the surface of cell membrane, but also penetrated inside the bacterial cells, cause formation of irregular-shaped pits and perforation on their surfaces and may also interact with the cellular macromolecules causing adverse effect including cell death. The data presented here are novel in that Al2O3 NPs are effective bactericidal agents regardless of the drug resistance mechanisms that confer importance to these bacteria as an emergent pathogen. Therefore, in depth studies regarding the interaction of Al2O3 NPs with cells, tissues, and organs as well as the optimum dose required to produce therapeutic effects need to be ascertained before we can expect a more meaningful role of the Al2O3 NPs in medical application.