The aim of the study was to analyze the evidence-based use of antibiotic therapy in the treatment of acute pancreatitis and to identify factors influencing the introduction of antibiotic therapy in the setting of transitional country clinical hospital. This retrospective study was conducted at Department of Internal Medicine at University Hospital Dubrava in Zagreb, Croatia. Data were collected from hospital records of patients treated for acute pancreatitis from January 1st, 2005 till December 31st, 2005. Data collected from patients' histories were compared with indications for antibiotic treatment and antibiotics with demonstrated therapeutic efficacy in acute pancreatitis which were obtained from published literature. Logistic regression was used to identify factors with a significant impact on deciding on the introduction of antibiotic therapy, and multivariate analysis was used to identify factors with a significant impact on the cost of treatment. Statistical significance was set at p < 0.05. Antibiotic therapy was used in 67.7% of patients with acute pancreatitis. Combination of amoxicillin plus clavulanic acid was most frequently administered, either as monotherapy or in combination with metronidazole and/or gentamicin (37.3%), followed by cefuroxime (32.8%) and cefoperazone (26.9%). The choice of antibiotic was appropriate in 35.8% of study patients; however in 29.9% of patients who were administered antibiotics had no indication for this therapy; and 46.9% of patients who had indications for receiving antibiotic therapy didn't receive it. In the groups of patients treated with antibiotics, the cost of treatment was significantly higher compared to groups of patients who were not treated with antibiotics (p = 0.0035; p = 0.0026). In addition to antibiotic therapy, the cost of treatment was significantly influenced by the length of hospital stay and treatment at intensive care unit. The use of antibiotics in the setting of transitional country university hospital in patients with acute pancreatitis is not evidence-based. Decision on the introduction of antibiotic therapy is not based on objective parameters of disease severity or evidence of therapeutic efficacy of particular antibiotics. The cost of treatment is significantly increased by the use of antibiotic therapy.