Background/Aim. Nosocomial infections (NI) are a serious health problem resulting in an enormous burden of excess morbidity and mortality rates, and health care costs. The aim of this study was to assess the prevalence of NI and to identify groups of patients at special risk for NI in the University Clinical Center, Kragujevac, Serbia. Methods. A period prevalence study design was used in this study. A survey of NI included all patients hospitalized in all departments in the University Clinical Center, Kragujevac. Results. Among 764 patients surveyed, the global prevalence rate of patients with at least one NI was 6.2% (95%CI = 5.6-6.8), while the prevalence of NI was 7.1%. The most frequent infections were surgical site infections (14.1%; 95%CI = 12.9-15.3), followed by pneumonia (2.3%; 95%CI = 2.1-2.5) in surgical patients. In medical wards, the most common NI were skin and subcutaneous tissue infections (1.6%l 95%CI = 1.4-1.8), and urinary infections (1.4%; 95%CI = 1.3-1.5). Overall, 85.1% NI were culture-proven; the leading pathogens were Pseudomonas species (40.0%), followed by Staphylococcus species (25.0%), Escherichia coli (22.5%), Proteus mirabilis (17.5%) and Klebsiella-Enterobacter (12.5%). Multivariate logistic regression analysis identified 3 risk factors independently associated with NI appearance: hospital stay ≥ 8 days (p = 0.0015), urinary catheter (p = 0.0022) and antibiotic use (p < 0.001). Conclusion. This study showed that NI are a serious health problem in our hospital. The most common infections were surgical site infections, followed by skin and subcutaneous tissue infection and urinary tract infections. Nosocomial infections were most common in patients in urological and orthopedic departments, and then in intensive care units. Prolonged hospital stay, urinary catheter and antibiotic exposure were risk factors independently associated with NI appearance.