Background: Surgical site infection (SSI) is the most common healthcare-associated infection following surgery and is associated with significant morbidity and mortality. Although, SSI prevention is complex and requires the integration of a range of measures before, during, and after surgery; up to 60% of these infections are estimated to be largely preventable by using evidence-based guidelines. Hence, as frontline caregivers, nurses can play a golden role in SSI prevention efforts by advancing their knowledge, attitudes, and practices toward the latest evidence-based preventive measures for SSI. Study aim: to evaluate the effect of evidence-based measures protocol on nurses' performance regarding the prevention of surgical site infection. Study Design: a quasi-experimental design. Setting: General surgery departments and surgical operating theater at Zagazig University Hospitals, Al Sharkia Governorate, Egypt. Subject: A convenience sample of all available nurses (70), who are working in the previously mentioned settings. Tools: A self-administered questionnaire to assess nurses' socio-demographic characteristics and knowledge; Likert scale to assess their attitudes towards the application of evidence-based preventive measures for SSI, and an observation checklist to assess nurses' practice of evidence-based SSI preventive measures. Results: Before the application of the evidence-based preventive measures protocol for surgical site infection, 10%, 43.3%, and 21.4%, respectively, of the nurses participating in the study had total knowledge, satisfactory practice, and positive attitudes towards evidence-based preventive measures for SSI, while after applying the protocol this percentage increased to reach to 74.3%, 88.6%, and 68.6%, respectively, with an improvement rate of 38% for knowledge, 16.89% for practice, and 20.65% for attitudes. Conclusions: Evidence-based measures protocol implementation had improved nurses' knowledge regarding evidence-based preventive measures of SSI, that in turn had shaped their positive attitudes toward the application of these measures, which results in improving their practices of evidence-based preventive measures of SSI, and this had ascertained by the Correlation matrix that had illustrated the presence of a statistically significant positive correlation of nurses' knowledge with their attitude and practice after the implementation of Evidence-based measures protocol. Recommendations: Evidence-based preventive measures of SSI should be incorporated into comprehensive surgical nursing quality improvement programs to improve patient safety.