Background: Adequate knowledge, attitude, and safe practice of infection prevention among healthcare providers are vital to prevent nosocomial infections. This study examined the level of knowledge, attitude, and practice of healthcare providers on infection prevention and its associated factors among health professionals working in Public Hospitals of Rwanda. Methods: Institution based cross-sectional study was conducted among 715 healthcare providers who were selected by convenient technique. Data were collected using interviewer-administered questionnaire. Multivariate logistic regression was performed to identify factors associated with practice of infection prevention and variables with a p-value < 0.05 were considered statistically significant. Results: More than half 70.8, 69, and 56.9 of healthcare providers had satisfactory knowledge positive attitude and satisfactory practice of infection prevention, respectively. Referral hospitals (AOR= 2.1, CI at 95% [= 1.5 – 2.9], p-value = 0.01] , Region (AOR = 1.4, CI at 95% [1.2 – 2.0], p – value = 0.04);(AOR = 1.54, CI at 95 % [1.01 -2.70], p – value < 0.01) , Education (AOR = 0.44, CI at 95%, [0.38 – 1.08], p-value 0.04) ; (AOR = 0.63, CI at 95% [0.37 – 0.87], p – value < 0.01) , attending a course (AOR = 1.54, CI at 95 % [1.02 – 1.68], p – value < 0.01), receiving HBV (AOR = 1.03, CI at 95% [0.49 – 1.12], p-value = 0.02) , working experience ≥ 10 years , and Positive overall attitude (AOR = 2.07, CI at 95% [1.01 – 3.42], p-value = 0.01) were positively associated with satisfactory practice of infection prevention. Conclusion: A considerable proportion of healthcare providers had satisfactory knowledge, attitude, and satisfactory practice of infection prevention. There is a need to improve training in health facilities.