Background Catheter-related urinary tract infection is an infection related to imported pathogens, which affects the urinary tract of patients with a urinary catheter. Catheter associated urinary tract infection is the most common nosocomial infection. Determining the catheter related urinary tract infections knowledge and practice of nurses, and the factors associated has paramount importance for improving the catheter-related urinary tract infection prevention practice, as well as efficiency and quality of care. Objective To assess the knowledge and practice of catheter-related urinary tract infection prevention and its associated factors among nurses working in public hospitals Oromia region, Ethiopia, 2021. Methods Institution-based cross-sectional study was conducted and to select 408 study participants a simple random sampling technique was used. A Structured self-administered questionnaire and observational check list were used to collect data. Descriptive and inferential analysis was done. Variable with p-value < 0.05 with 95% CI was declared statistically significant. Result From the distributed questionnaires 408 participants returned with their response which gives 96.7% response rate. Out of respondents, 63.5%, had adequate knowledge, but only 34.6% of study participants have good practice on Catheter-related urinary tract infection prevention. Having in-service training on infection prevention, working in intensive care unit and having access to infection prevention guide line were significantly associated with both the nurses’ adequate knowledge and good practice of catheter-related urinary tract infection prevention while age and year of experience, were factors significantly associated with nurses good practice of catheter-related urinary tract infection prevention. Conclusions The finding of this study revealed that more than half nurses have adequate knowledge on catheter-related infection prevention, but around one-third of them had good practice, which suggests difference between knowledge and practice. In-service training on infection prevention, the current unit of working, and the presence of a guide line were significantly associated with both the knowledge and practice of nurses toward catheter-related urinary tract infection prevention. There is a need of improving knowledge and practice of nurses towards catheter related urinary tract infection prevention through in-service training and use of guideline at working units.