Purpose: This study aimed to identify the factors influencing infection-related characteristics and patient safety culture on awareness of blood-borne infection prevention between operating room nurses and general ward nurses. Methods: Participants were 198 nurses(operating room nurses 98 and general ward nurses: 100) working at three general hospitals and three university hospitals in three cities. Data were collected using a structured questionnaire from September 11 to October 14, 2020. Data were analyzed using descriptive statistics, t-tests, ANOVA, Pearson’s correlation coefficient, and multiple regression with IBM SPSS/WIN 26.0 program. Results: Typically, 39.8% of nurses in the operating room and 24.0% of ward nurses experienced injuries such as needles and sharp instruments used by the patient. The awareness of patient safety culture was identified to be higher for the ward nurses. Factors influencing the awareness of blood-borne infection prevention in operating room nurses were patient safety culture and wearing protective equipment for infection prevention while nursing infected patients. Moreover, the explanatory power of these variables was 19.4%. In general ward nurses, the patient safety culture was identified as a significant predictor, which accounted for 16.5% of awareness of blood-borne infection prevention. Conclusion: To prevent hospital infection, a strategy is needed to improve the level of awareness of blood-borne infection prevention and patient safety culture of operating room nurses. To this end, the difference in infection-related characteristics and influencing factors between the operating room nurses and the general ward nurses should be considered and planned.