Background: Standard isolation precautions are designed to reduce the risk of acquiring occupational infections from both known and unknown sources in the healthcare setting. Awareness and compliance with these recommendations is crucial for the prevention of occupational nosocomial infections in healthcare workers. Improving the compliance of standard precautions (SP) is one of the most important ways to reduce healthcare associated infections, but also the most difficult to achieve. The aim of this study was to evaluate the levels of knowledge, attitude and practice of surgeons and surgical residents in Isfahan, regarding standard precautions.Methods: In a cross-sectional, 140 surgeons and surgical residents were evaluated about standard precaution in Isfahan University of Medical Sciences. Questionnaires were handed to participants who were willing to take part in the survey. The questionnaire included demographic data and also knowledge, attitude and practice of standard precaution. The questionnaire was self-administered. Descriptive and inferential statistics including Spearman and Mann-Withney tests were performed.Finding: In this study, 91 residents and 48 surgeons were assessed with the mean age of 38.26 ± 10.86 years. The mean for surgeons’ knowledge was 8.25 ± 1.22 and mean for surgical residents’ knowledge was 7.94 ± 1.37 (P > 0.05). The mean for surgeons’ attitude was 40.56 ± 3.46 and mean for residents’ was 38.68 ± 3.70 (P < 0.05). The mean of surgeons’ practice was 4.78 ± 2.29 and mean of surgical residents’ practice was 3.80 ± 2.21 (P < 0.05).Conclusion: The results suggest that among study groups, the levels of knowledge and attitude regarding standard isolation precautions were acceptable; although compliance level was moderate. Therfore, for better acting attitude and practice, we have to educate and update healthcare worker especially who had poor practice.