Introduction: Health care-associated infections (HCAIs) poses a real and serious threat to both the patients and health care workers. A significant number of patients acquired health care associated infections worldwide, and this has devastating effect on both the patient and the health system. It is estimated that more than 1.4 million people worldwide are suffering from infections acquired in hospitals. Even though infection prevention plays a key role in preventing and reducing the rate of healthcare associated infection, little is known about current staffing and structure of infection prevention and control programs. Objective: To assess Infection prevention practice and associated factors among healthcare providers in Bishoftu Referral Hospital south east Ethiopia from Dec 4, 2019 to Dec 20, 2019 G.C. Methodology: Institution based crosssectional study was conducted to assess practice towards infection prevention and associated factors in Bishoftu Referral Hospital. The data was collected using structured self-administered questionnaire and supported by an observational check list. Infection prevention practice was calculated using 22 items and median was used a cut-off point to generate a binary practice outcome. The data was entered into EPI info 7 and then exported to SPSS version 20 for data management and analysis. Bivariate and Multivariable logistic regression analysis was carried to assess significance of determinants. Results: One hundred fifty eight (158) health care professionals were included in the study. This study showed that 60.4% of Health Care Providers had safe infection prevention Practice (95% CI (51.9%, 68.2%). Among the determinant factors, working in emergency ward [AOR=4.327, 95% CI (0.412, 45.464)], knowing the presence of infection prevention committee [AOR=7.629, 95% CI (1.580, 36.831)] and being a midwife [AOR=16.39, 95% CI (1.074, 250.171)] were significantly associated with safe infection prevention practice. Conclusion and Recommendation: The findings of this study show that around 40% of healthcare professional didn't adhere to safe infection prevention. Working wards, infection prevention committee and profession were factors significantly associated with infection prevention practice. The hospital should give emphasis for all working wards to increase adherence to infection prevention practice.