Background: In the past 20 years, the overall incidence of health care associated infections has increased by 36%. It is estimated that more than 1.4 million people worldwide are suffering from infections acquired in hospitals. Sub Saharan Africa harbors the largest population of people living with infectious disease mainly with HIV/ AIDS including Ethiopia.

Methodology: Institution-based cross sectional study was employed; and data was collected using self-administered questionnaires. Data analysis was carried out manually by using tally and calculator, and descriptive summaries were used to describe the study variables like frequency tables and charts.

Result: 50.55% of health care workers had good knowledge; 69.23% had positive attitude and 48.35% had safe practice of infection prevention. Above half (51.65%) of them had unsafe practice of infection prevention.

Conclusion: Health care workers’ knowledge, attitude and practice towards infection prevention in Dubti Referral Hospital were not sufficient, favorable and safe enough to the expected standard. There were gaps regarding infection prevention practice like proper utilization of personal protective equipments.