Objective: The aim of the study was to investigate nurses' knowledge regarding the prevention of surgical site infection (SSI), and to examine the relationship between nurses' demographic characteristics and educational level and their level of knowledge in prevention of SSIs. A further aim was to examine the differences in nurses' knowledge with respect to selected variables and to identify the most significant predictors of nurses' knowledge regarding the prevention of SSIs, to support the provision of high-quality nursing care. Method: A prospective, observational study of a convenience sample of nurses and assistant nurses working in surgical departments, in a public general hospital for adults in Attica, during May to August 2016. For data collection, an anonymous self-completion questionnaire was developed and tested for comprehension and acceptability. Results: Data was collected from 148 nurses and assistant nurses, 121 (81.8%) were female, 73.6% were aged 36–50 years and 43.9% had 11–20 years of experience. With regards to educational level, 66.2% had a degree from a technological educational institute and 10.1% had a Master's degree. Furthermore, 18.2% had a surgical specialty and 59.5% had received special training on surgical infections. The majority of respondents did not chose the correct definition of the time of occurrence of SSIs. Several statistically significant correlations were observed between knowledge on safer hair removal and respndent age (p=0.037), educational level (p=0.003), professional experience (p=0.048), and training in SSIs (p=0.009). Conclusion: The results of this study revealed that the majority respondents had a high level of knowledge regarding the prevention of SSIs, which contrasted with a low level of knowledge regarding their full definition of the time of occurrence.