ABSTRACT Background: Infection prevention practice is a serious concern for health care workers; it is a major risk for the transmission of infections in the hospital. Infection prevention practice is low and not well explored in developing countries. Therefore, the objective of this study was to determine the level of infection prevention practice and asses associated factors among health care worker at Tibebe Ghion Specialized Hospital, Bahir dar university, Amhara region, Ethiopia, August 2020. Methods and Materials: Institution based cross-sectional study was conducted among healthcare workers in Tibebe ghion specialized hospital, Bahir dar university. Data was collected from August 2020 to January 2020. A total of 257 healthcare workers was included in the study. Proportional random sampling technique was employed to select the study participants. The collected data was cleaned, coded and entered and analyzed using IBM SPSS statistics data editor version 25. Multivariable binary logistic regression analysis was conducted. P <0.05 will considered as statically significant. Results: Two hundred fifty-seven healthcare workers participated in the study. One hundred sixty-nine (65.8%) health care workers had good infection prevention practices. Having positive attitude towards infection prevention practices (AOR=4.44, 95%CI: 1.97-9.99), having adequate PPE (AOR=2.68, 95%CI: (1.28-5.63), having training on IP (AOR = 3.59, 95%CI: (1.64-7.83), availability of safety manuals (AOR = 2.23, 95%CI: (1.15-4.33) and presence of adequate water supply (AOR=3.49, 95%CI: (1.78-6.84) were predictors of good infection prevention practices. Conclusion: Two-third of the healthcare workers had good infection prevention practices. Having positive attitude towards infection prevention practices, having adequate PPE, having training on IP, availability of safety manuals and presence of adequate water supply were predictors of good infection prevention practices. To sustain good practices, adequate pre-service and in-service training should be in place to equip and update health care workers about infection prevention precautions. The need for continuous supervision should be implemented to strengthen adherence for infection prevention practices among workers along with sustainable and reliable water supply is crucial. Key words: Healthcare workers, Infection prevention and control, hospital, Ethiopia