OBJECTIVE: To analyze antibiotic use in a tertiary care hospital in Mexico. MATERIALS AND METHODS: We conducted two point prevalence surveys based on the World Health Organization methodology in a tertiary care hospital in Guadalajara, Mexico. We surveyed the clinical records of patients with active antibiotic prescriptions (APs) in medical (MED), surgical (SUR), medical-surgical (MIX) wards, and intensive care units (ICUs). Descriptive statistics were estimated using Stata software. RESULTS: We analyzed 929 APs from 403 patients. The prevalence of antibiotic use in the hospital was 47.5%. Antibiotics were more used in ICUs (59.5%) and MIX wards (54.8%). The main reasons for antibiotic use were community-acquired infections (45.2%), and preoperative prophylaxis (23.1%), mostly multidose and prolonged (89.3%). APs were mainly empirical (92.4%), administered parenterally (95.9%) and lacked subsequent review (30.3%). Bacterial culture testing was limited (30.5%). The most used antibiotics were ceftriaxone (18.9%), clindamycin (8.5%), and meropenem (8.2%). Most APs corresponded to Access (56.4%) and Watch antibiotics (35.6%) (AWaRe, WHO). CONCLUSIONS: We revealed frequent prescribing patterns of broad-spectrum antibiotics and differences in their use possibly related to patients' clinical profiles.