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on human health. Several infections caused by this bacterium occur in both community acquired and hospital-acquired settings. Objective: This research aims to study the effect of magnesium oxide nanoparticles biosynthesized from Bacillus subtilis against multidrug-resistant (MDR) S. aureus isolated from various clinical infections. Methodology: A total of 230 specimens were collected from patients with different clinical infections referred to hospitals in Al-Najaf Province, Iraq, representing various age groups and both genders. Standard microbiological assays were used to identify S. aureus isolates, which were then subjected to antimicrobial susceptibility testing against 18 types of antibiotics using the disc diffusion method. Magnesium oxide nanoparticles (MgO NPs) were synthesized by mixing B. subtilis culture filtrate with magnesium nitrate solution. The color change from pale yellow to white indicated the formation of MgO NPs. Results: Out of the 230 specimens, 80 were identified as S. aureus isolates and tested for antimicrobial susceptibility. The results showed the highest resistance rate to Penicillin G (97.5%), while S. aureus showed no resistance to Vancomycin (0%). Among the 80 isolates, 45 (56.25%) were identified as multidrug-resistant (MDR S. aureus).The antibacterial activity of MgO nanoparticles at different concentrations (100, 200, 300, and 400 μg/ml) against S. aureus demonstrated that the inhibition zones increased proportionally with the concentration of MgO nanoparticles. Conclusion: This study concludes that higher concentrations of MgO nanoparticles biosynthesized by B. subtilis lead to an increased inhibition zone against MDR S. aureus, indicating a concentration dependent antibacterial effect. INTRODUCTION Staphylococcus aureus is a major human pathogen capable of adapting to diverse hosts and environmental conditions, causing a wide range of infections. Additionally, it is one of the leading causes of both hospital-acquired and community-acquired infections. It can cause infections of the bloodstream, skin and soft tissues, and lower respiratory tract; infections associated with medical instrumentation such as central-line associated bloodstream infections; as well as serious deep-seated infections such as osteomyelitis and endocarditis¹. S. aureus is among the most common bacterial pathogens in humans and is responsible for multiple infections, including infective endocarditis, skin and soft tissue infections (e.g., impetigo, folliculitis, furuncles, carbuncles, cellulitis, and scalded skin syndrome), septic arthritis, gastroenteritis, meningitis, urinary tract infections, pneumonia, and bloodstream infections².