Purpose: Hospital acquired infection (HAI) is an infection acquired a major global concern of wellbeing, affecting the quality of care in the health care setting. Routine surveillance of infection is an important part of infection prevention (IP) and quality assurance in hospitals. The objective of this study was to determine the prevalence and associated factors of HAI at inpatients, in Hawassa university comprehensive specialized hospital (HUCSH). Method and materials: A weekly based cross-sectional study was conducted at inpatients in HUCSH. All eligible inpatients admitted for at least three days on the day of survey were included. The study was conducted from June 28/2021 to August 23/2021. Environmental health professionals and nurses collected the data according to the Centers for Disease Control through observational assessment documented by physician. Coded and cleaned data from Epi-data version 3.5 were transferred to SPSS version 23 for analysis. Univariate and multivariable logistic regression analyses were used to determine the prevalence of HAIs and associations between independent and dependent variables. . Results: A total of 413 patients were included in this survey, the median age of the participants was 26 years (interquartile range:3 month to 80 years). A total of 352 (85.2%) patients diagnosed with non-fatal disease during the survey. There were 49 patients with HAI, with a mean prevalence of 11.9 %. Coagulate negative staphylococcus (CONS) 27.27%), and E. coli (27.27) were the most frequently reported HAI-causing pathogens from the result of culture. The association of patients admission diagnosis (admit with rapidly fatal disease and ultimately fatal disease), length of hospital stay (more than five days of hospital stay) and absence of running tap water at patients room with the occurrence of HAI were statistically significant. Conclusion: Surgical site infections (SSI) and blood stream infection (BSI) were the most common types of HAIs. The proportion of HAIs among wards, indicated a large variability. Therefore, Hospital management and health care workers (HCWs) should give more attention on the practice of infection prevention (IP) in order to achieve a reduced prevalence of HAIs in the study setting.