Background: Methicillin-resistant Staphylococcus aureus (MRSA) infection is considered one of the nosocomial infections that can infect patients and healthcare workers (HCWs) and negatively affect the quality of care provided in the hospital. Objective: Evaluate the knowledge of HCWs regarding the prevention of MRSA infection in Thi-Qar Governorate. Methods: A descriptive cross-sectional study was conducted for 362 HCWs randomly selected from four hospitals and distributed as follows: 125 from Nasiriyah Teaching Hospital, 80 from Al-Hussein Teaching Hospital, 80 from Al-Haboubi Teaching Hospital and 77 from Souk Al-Shuyoukh General Hospital during the period from October 1 (2022) to May 1 (2023) and data was collected by using self-reported paper-based questionnaires. Results: Our study showed 68.8% of HCWs enjoyed a moderate level of knowledge, and there was a strong correlation (P value <0.05), between knowledge and some sociodemographic and occupational characteristics of the participants, which include age, educational level, job title, years of service and workplace in the hospital. Additionally, a relationship between knowledge and the source of the MRAS information was demonstrated which is a highly significant association between the total knowledge score and the sources of information. Conclusions: The knowledge of MRSA infection prevention among HCWs was moderate.