Pathogenic strains frequently spread infections by producing virulence factors like potent protein toxins and the expression of a cell-surface protein that binds and inactivates antibodies. Staphylococcus aureus is one of the leading pathogens for deaths linked to antimicrobial resistance and the emergence of antibiotic-resistant strains. There is currently no licenced S. aureus vaccine, despite extensive research and development. Therefore, the aim of this study includes biosynthesize of Nickel oxide-nanoparticles (NiO-NPs) using Lepidium sativum aqueous plant leaves extract, and use of the biosynthesized nanoparticles as antibacterial and anti-biofilm against multidrug resistant S. aureus