Background: Surgical Site Infection (SSI) is one of the most common healthcare-associated infections, its account for up to 16% of all healthcare-associated infections worldwide. The SSIs can contribute to post-operative morbidity, prolonged recovery, delayed discharge and increasing cost. Nurses' knowledge of the evidence-based recommendations is necessary to provide high-quality nursing care. Aims: To assess the level of Jordanian nurses' knowledge regarding evidence based guidelines for the prevention of SSIs, to describe the relationship between nurses' knowledge and selected sociodemographics, to examine the differences in nurses' knowledge with respect to selected dichotomized variables, and to identify the most significant predictors of Jordanian nurses' knowledge regarding evidence based guidelines for the prevention of SSIs. Design: Cross-sectional design. Sample: Two hundred registered nurses at four targeted hospitals were recruited conveniently. Results: The mean of the total knowledge scores of the sample was 3.28 out of nine (SD = 1.72, range = 0 -7), the median was 3 out of nine. There were a statistically significant correlation between all tested continuous sociodemographics variables and the total knowledge score (p > 0.05). There is a significant difference in nurses' knowledge between those who are attending to special surgical related training course and who are not. The most significant predictors of Jordanian nurses' knowledge were: the number of credit hours attended by nurses for surgical-related training courses and the total years of work experience in nursing. These variables explained 16.7% of variance. Conclusion: The results of this study shed light on the obstacles that hampers the Jordanian nurses' knowledge regarding evidence based guidelines for the prevention of SSIs in acute care settings. However, the successful implementation of infection control measures, particularly SSIs prevention measures, and well-structured continuing education programs are considered as a substantial element that would improve nurses' knowledge.