Background: Hospital-acquired infections are a major complication of hospital treatment. The growing presence of multidrug-resistant pathogens contributes to increased mortality and costs, particularly in intensive care units where patients are predisposed to numerous risk factors. Comprehensive data about hospital-acquired infections from Serbian intensive care units is scarce. The aim of this study was to determine the presence of hospital-acquired infections among intensive care unit patients and look into the patterns of antimicrobial resistance, risk factors, and incremental costs of diagnosis and antimicrobial treatment. Methods: This retrospective study included 355 patients over a two-year period. Etiology, antimicrobial resistance patterns, and incremental costs of diagnosis and antimicrobial treatment were examined. Risk factors for infection acquisition, as well as length of stay, were statistically analyzed using Pearson’s chi-square tests and logistic regression analysis. Results: At least one hospital-acquired infection was identified in 32.7% of patients. A total of 204 infection episodes were documented, the most common type being urinary tract infections (36.3%). Clostridium difficile , Klebsiella spp. , and Acinetobacter baumanii were the most common isolates. Antimicrobial resistance rates < 20% were observed for linezolid (0%), colistin (9%), and tigecycline (14%). Resistance rates > 50% were seen in all other tested antibiotic agents. Mortality rates were not higher in patients who acquired only one hospital-acquired infection (p=0.09), but were significantly higher for patients in whom more than one episode occurred (p=0.038). Length of stay > 20 days carried a 7.5-fold increase in odds of acquiring an infection (CI 4.4-12.7, p<0.001), whereas length of stay > 30 days carried a 10-fold increase (CI 5.5-16.1, p<0.001). During the study period, over 37,000 EUR was incrementally spent on diagnosis and antimicrobial treatment for hospital-acquired infections. Conclusion: Our results suggest a high prevalence of hospital-acquired infections and very high antimicrobial resistance rates compared to most European countries. Together with the first published results regarding incremental costs from Serbia, our observations require large-scale prospective follow-up studies in order to obtain a deeper insight into the actual burden of hospital-acquired infections.