Background: The key to the effective management of healthcare wastes is segregation of the waste at the point of generation; no matter what final strategy for treatment and disposal of wastes is selected, it is critical that waste streams are separated. In Ethiopia, health care waste segregation practice among healthcare workers is overlooked and scarcely addressed in the scientific literature. Objective: to assess the practice of infectious waste segregation and its associated factors among health care workers in hospitals in Dire Dawa, hospitals, Eastern Ethiopia, 2020. Methods: Institution based cross-sectional study was conducted from Oct 01 up to Nov 30, 2020 in two randomly selected hospitals from public and private. A total of 280 study participants were involved using stratified simple random sampling technique by professional categories. Data was collected through self-administered structured questionnaires and observation checklist. It was analyzed by using SPSS version 25. Bivariate and multivariable logistic regression model were used to identify factors associated with practice of health care workers. Results: A total of 280 participants were involved in this study, 228 (81.4%) and 52 (18.6%) were from public and private respectively. The overall mean age of the respondents was 33.26 (±6.65) years and majority 173 (61.8%) were female, 200 (71.4%) were married, 250 (89.3%) were found to have first degree, more than half 184 (65.7%) were Nurses and the mean working hours per week was 44.07 hours (±18.843). About 158 (56.4%) of the respondents had good practice. Hospital ownership AOR 0.088 CI 95% (0.033-0.235), working hours [(AOR = 4.275, 95% CI: (2.107-8.675)], adequate knowledge [(AOR = 2.424, 95% CI: (1.274-4.612)], training on IPC [(AOR = 2.74, 95% CI: (1.150-6.527)], training on waste segregation (AOR 3.918 CI 95% (1.514-10.138)), availability of rules and regulations on HCWM [(AOR = 8.212, 95% CI: (3.844-17.545)}, and availability of color coded waste bin [(AOR = 9.53, 95% CI: (4.516-20.10)] had statistically significant association with waste segregation practice. Conclusion: Generally, the practice of HCPs on infectious waste segregation was poor, training on IPC and waste segregation, availability of waste management policy, PPE, color coded waste bin, working hours, knowledge and attitude were significantly associated with the practice.