Background: Medical literature reports that adequate prophylaxis with antibiotics can reduce the incidence of postoperative infections. Objective: To investigate the impact of adopting multiple practices for improving the rational use of antibiotic prophylaxis in patients undergoing cholecystectomy and evaluate, during 2 periods, compliance with the hospital Drug Committee's recommendation and guidelines published in the medical literature. Methods: Data were collected from patients' medical records at the Hospital de Clíniacas de Porto Alegre (HCPA), Brazil, in 2003. We evaluated 222 procedures as set by guidelines published in the literature and the hospital's recommendation. Results: In 24.5% of the cases, the choice of whether to use the prophylactic antibiotic was not made according to the guidelines. The choice of the antibiotic agent, prophylaxis duration, dose administered, and time for administration was appropriate in 95%, 80.5%, 100%, and 81.8% of cases, respectively. A significant improvement in guideline compliance was observed since multiple practices were adopted to improve the rational use of antibiotic prophylaxis. Conclusions: Compliance with the guidelines for antibiotic prophylaxis for cholecystectomy, in combination with a greater integration among the professionals involved in the HCPA Infection Control Service, resulted in a more appropriate use of these prophylactic agents. Improving physicians' awareness of the importance of meeting guidelines is critical and will benefit patients and the hospital as a whole.