Background: Infection prevention is universally acknowledged as a vital component of comprehensive approach to patient and healthcare worker safety, quality improvement, and improved health outcomes. Infections acquired during health-care delivery are a significant public health problem around the world. Thus, infection prevention is not only the most cost-effective option, but also the best strategy to prevent the spread of disease within health facilities. Therefore, the objective of this study was to assess infection prevention practices and associated factors among health care workers in governmental health care facilities in South Gondar zone, North West Ethiopia. Methods: Institutional based cross-sectional study design was conducted from 15 February-15 April, 2020 in South Gondar zone among healthcare workers.Multistage was usedas sampling procedures. Data were collected using pre-tested self-administered structured questionnaire. Data were entered and analyzed using Epi-data version 3.1 and SPSS version 23 respectively. The model was fitted using multivariable logistic regression to identify associated factors with infection prevention practices. Odds ration with a 95% confidence interval was used as measure of association. Finally p-value less than 0.05 were used to declare statistical significance. Results: Six hundred two (97%) healthcare workers participated in the study. Three hundred thirty six 60.8%(57.1-64.8%) 95%CI health care workers had good infection prevention practices. Availability of gloves, AOR 3.69(2.015, 6.789)95%CI, Availability Safety box, AOR=1.9(1.2, 3.1)95% CI, Presence of Infection prevention guide line, AOR=0.498(.32, .83) 95% CI, presence of alcohol swab AOR = 0.49(.29, 82)95% CI. Accessibility of hand washing items AOR=2.0(1.2, 3.4) 95% CI were found to be significantly associated. Conclusion: Nearly two-third of the healthcare workers had good infection prevention practices. On infection prevention measures, availability of guide lines, safety box, gloves, alcohol swab, and accessibility of items for hand washings were predictors of good infection prevention practices. To sustain good practices, accessible of hygiene materials should be kept to all units of health facility all times.