Background: To avoid the spread of communicable diseases, infection control and preventive measures are essential in all health care settings. Every health care professional, especially nurses, plays an important role in reducing the risk of health care-associated infections. Our goal is to examine the knowledge and attitudes regarding infection control and prevention measures. Methods:  Cross sectional study was used to achieve the aim of the study. Convenience sample was used to involve 45 nurses at King Khalid Hospital and New Najran General Hospital in Najran city, using a self-administered questionnaire to examine the knowledge and attitude of nurses regarding infection control and prevention measures. Results: According to the study, 76.7% of the individuals had good knowledge, while 23.3% had poor knowledge. The majority of participants (78.8%) had a positive attitude toward infection control and prevention measures, whereas 21.2% had a negative attitude. There were statistically significant variations based on total years of experience, gender, education level, and the occurrence of needle stick during years of employment. However, due to the years of experience in the surgical wards, there were statistically significant variances. Conclusion: The findings of this study highlight a generally positive trend in knowledge and attitudes toward infection control and prevention measures among healthcare professionals. A substantial majority, comprising 3 quarter, demonstrated good knowledge, emphasizing a foundational understanding of essential practices. Additionally, a notable 78.8% expressed a positive attitude, indicating a prevailing commitment to infection control. The identified statistically significant variations based on total years of experience, gender, education level, and needle stick occurrences offer valuable insights. Recommendation: Develop targeted educational initiatives focusing on individuals with poor knowledge and negative attitudes, customizing content based on specific demographic factors such as gender, education level, and years of experience.