This study aimed to estimate the prevalence and risk factors for health-care-associated infection (HAI) in all 9 hospitals of the central-east area of Tunisia in 2005. Of 1373 patients admitted for more than 48 hours, 74 developed HAI, a prevalence of 5.4% (95% CI 4.2%–6.6%). The prevalence was significantly higher in the intensive care units (18.4%) and neonatal departments (12.7%). There were 79 infections and the most frequent sites of infection were respiratory tract and urinary tract. Microbiological examination was performed for 25 cases of HAI and Pseudomonas aeruginosa was identified in 8 cases. Multiple logistic regression analysis indicated that HAI was linked to diabetes (OR = 2.0), immunosuppression (OR = 3.3), length of stay (OR = 4.5), central venous catheter (OR = 2.5) and peripheral venous catheter (OR = 10.2). We conclude that HAIs are of concern in this area of Tunisia.