Introduction: Catheter-Associated Urinary Tract Infections have been highly associated with patient discomfort, increased hospitalization and healthcare costs, morbidity and mortality. Lack of knowledge and practice among nurses regarding basic infection control protocols could lead to hospital-acquired infections. Therefore, this study aimed to assess knowledge, practice and associated factors of Catheter-Associated Urinary Tract Infections prevention among nurses working at referral hospitals in the West Oromia region, Ethiopia, 2022. Methods: A facility-based cross-sectional study was conducted among 423 nurses at five referral hospitals of the West Oromia region from May1-20, 2022. A simple random sampling technique was used to select nurses. A pre-tested and structured self-administered questionnaire was used to collect the data, and then coded and entered into EPI- Data version 4.6; cleaned and analyzed using Stata version 16.0. All variables with P-value < 0.25 in the binary logistic regression analyses were included in the multivariable regression analysis. The degree of the association was interpreted by using the adjusted odds ratio with 95% confidence intervals, and the significance level was declared at P-value < 0.05. The Hosmer–Lemeshow test was checked for the fitness of the model. Results: Among 423 nurses, 411(97.2%) nurses participated. This study found that the knowledge and practice of nurses toward preventing Catheter-Associated Urinary Tract Infections was 57.9% with 95% CI (53.1, 62.6) and 54.5% with 95% CI (49.6, 59.3) respectively. Good knowledge was significantly associated with working experience [AOR = 2.8, 95% CI (1.5, 5)], training [AOR = 2.1, 95% CI (1.04, 4.2)] and had guideline [AOR = 2.6, 95% CI (1.3, 5.4)]. Good practice was significantly associated with the work experience [AOR = 2, 95%CI (1.05, 4.05)], guideline [AOR = 2.3, 95% CI (1.02, 5.2)], training [AOR = 2.3, 95% CI (1.06, 5.1)] and good knowledge [AOR = 8.7, 95% CI (5.1, 14.8)]. Conclusion: This study showed that more than half of nurses had good knowledge and good practice regarding preventing catheter-associated urinary tract infections, and were significantly associated with work experience, training and guideline.