Abstract Background: Hospital acquired infection is the most common cause of morbidity and mortality for hospitalized patients. Ninety percent of hospital acquired infections are due to bacterial infection. Assessing the rate of hospital acquired infection will be helpful for appropriate interventions that will reduce mortality, morbidity and higher treatment expenditure. Objective: This study was aimed to assess the magnitude and antimicrobial resistance profile of hospital acquired bacterial infection among patients admitted in different wards of Debre Markos Referral hospital. Methods: Hospital based cross-sectional study was conducted among patients admitted in different wards of DMRH from April, 2018 to July, 2018. Data was collected using a structured questionnaire via face to face interview and patient card review. A total of 365 patients with presumptive of HAI were included using convenient sampling technique. Clinical specimens were processed in to appropriate culture media and organism isolated using Gram staining and biochemical test. Antimicrobial susceptibility test was performed for isolates based on Kirby Bauer Disk diffusion technique. Data was coded, entered and analyzed with SPSS version 23.Odds Ratio (OR), p-value and their 95% Confidence Intervals (CI) were calculated and the result was considered statistically significant at P < 0. 05. Result: The magnitude of hospital acquired bacterial infection at DMRH was 78(21.4%). Staphylococcus aureus 28(7.7%) and E. coli 12(3.3%) organism were the most common agents of hospital acquired infection. Multidrug resistance rate of isolates were 52.3% and Pseudomonas aeruginosa had 63.4% rate of multidrug resistance. Patients hospitalized for ≥5 days, admitted on unclean bed sheet, irregular implementation of aseptic procedure in patients care, urinary catheterization, and surgery were significantly associated with hospital acquired bacterial infection. Conclusions and Recommendations: The magnitude of HABI rate was high in the study area. The bacterial isolates from patients were also highly resistant for commonly prescribed antimicrobial drugs. Great effort should be carried out to minimize Hospital acquired infection and multi drug resistance. Key words: Hospital acquired infection, DMRH, MDR, Ethiopia.