Background: Adherence to infection prevention and control guidelines is critical to improving the quality of hospital care based on their efficacy in reducing the occurrence of infections that compromise patients' outcome. Nurses are front line of protecting themselves and clients from infectious disease. Nurses perform clinical procedures or other activities that can generate waste and expose themselves and clients to potentially infectious disease. This put nurses to be at higher risk for acquiring blood borne pathogens at health care facilities. Objective:The main objective of this study was to assess the level of knowledge and practice on adherence of infection prevention and its associated factors among nurses in Jimma University medical center.Method: Cross-sectional study design was conducted from April 1 to 10, 2016 and systematic sampling technique was used to select a total of 231 study subjects and data were collected using self-administered questionnaire and entered to Epi data manager and client analyzed using SPSS version 20 software and the analyzed data was presented using graphs, charts and tables were used to summarize and present major findings. Result:The overall knowledge of nurses is 83.08%. Majority of the respondents 215 (83.08%) was knowledgeable regarding infection prevention and 16 (16.02%) was less knowledgeable. The overall practice of respondents were 61.08% and 148 (64.06%) of nurses has good practice but 83 (35.09%) has less practice. Conclusion and recommendation:Majority of the respondents were knowledgeable regarding infection prevention principles. Although there were some gaps regarding infection prevention practice like washing hands in between patients, wearing of gloves when giving patient care, wearing of masks and goggles, recapping of used needles. The nurses were not strictly adhered to infection prevention practices due to insufficient supply of resources, working experience, negligence and shortage of time.