Staphylococcus aureus (S. aureus) remains one of the most frequently occurring community-acquired as well as hospital-acquired pathogens with high rates of hospital-acquired infections.  S.aureus is an important pathogenic bacterium. It resides predominantly in the anterior nares, and extra-nasal sites including the skin, perineum, and pharynx, and less frequent in the gastrointestinal tract and the vagina. About 30% of the general population is nasal carriers of the bacterium. Infections caused by this organism could be exogenous or endogenous in origin, and they include impetigo, cellulitis, osteomyelitis, endocarditis, toxic shock syndrome (TSS), septicemia, and pneumonia.  Healthcare-associated infections by S. aureus are about 20% worldwide. S. aureus infection at NICU remains a major health challenge as colonized healthcare workers, patients, and contaminated surfaces serve as reservoir for infection. The incidence of healthcare-associated infections caused by S. aureus continues to increase worldwide. The goal of this study is to assess the nasal carriage of S. aureus isolated from neonates and health care workers (HCWs) in the NICU of Beni Suef University Hospital. Also, our study provides valuable insights documenting important factors that affect susceptibility and outcomes of neonatal sepsis which is a global public health issue.