Objective: To determine the point prevalence and characteristics of antibiotic use in different hospitalization areas of the Hospital Nacional Dos de Mayo. Materials and methods: We carried out a 3-week cross-sectional study with a census approach (using methodology from the World Health Organization) to determine the point prevalence of antibiotic use in inpatients from hospitalization areas, regardless of whether or not they were on an antibiotic regimen. Results: We identified 358 patients, with a mean age of 49 (± 25.3) years, predominantly adults (88%). Antibiotics were used in 51.7% of the hospitalized patients; only 57.3% of the prescriptions followed a regimen based on clinical practice guidelines for antibiotic treatment, while 28.5% did not follow any standardized recommendation. Therapies were empirical in 86.8% and directed in 13.2% of the cases. Conclusions: Antibiotics were used in more than 50% of hospitalized patients. However, about one third of the prescriptions were not based on any clinical practice guidelines. Despite the existence of an antimicrobial control program in the hospital, appropriate antimicrobial use still needs to be optimized.