Introduction: Contact of health care workers and patient at health facility lead to the transmission of pathogenic organisms to each other. Hospital acquired infection prevention is standardized guide lines contain multitude protocols needed to be implemented by health care workers to reduce hospital acquired infections.Objective: To assess health care workers knowledge, attitude and practice towards hospital acquired infection prevention at Dessie referral hospital. Method:Institutional based cross sectional study was conducted among health care workers. The subjects were selected by proportional allocation in each respective department and simple random sampling method was employed. Statistical package for social sciences (SPSS) was used to analyze the data and presented in frequency tables and graph.Results: 191 study subjects were participated in the study which gives a response rate of 90.5% from 211 total sample sizes. The study subjects had given their response according to self-administered questioner. The result indicates that 86.4%, 76.4% and 77% of respondents had good knowledge and favorable attitude and poor practice towards hospital acquired infection prevention respectively. Conclusion:Even though the majority of the health care workers had good knowledge and favorable attitude, more than two third of them had poor practice towards hospital acquired infection prevention. Therefore health workers should strictly follow hospital acquired infection prevention guidelines. Improving sustainable supplies like personal protective equipment, water supply and hand washing facilities at patient care site is vital to correct the poor practice of infection prevention.