In 2018, the WHO published a methodology for conducting a point prevalence survey (PPS) of antibiotic use in hospitals. The aim of this study is to report the use of antibiotics in six second-level hospitals in Mexico using this methodology. Methods: A multicenter cross-sectional study based on the 2021–2023 adaptation for Latin American hospitals was conducted in internal medicine, surgery, intensive care unit (ICU), obstetrics and gynecology and pediatrics departments of the IMSS in the western region of the state of Mexico. Results: The overall prevalence of antibiotic use was 61%; the services with the highest prevalence of prescription were general surgery (79%) and the ICU (78%). A total of 846 patients were surveyed; there were no differences in antibiotic use or non-use in terms of gender, surgical procedure and invasive devices, but there were differences in median age and comorbidities. Adherence to guidelines was 53.9%. The three main antibiotics used were third-generation cephalosporins (28%), carbapenems (13%) and glycopeptides (9%); for the type of indication, for CAI and prophylaxis, the rates of use of third-generation cephalosporins were 29.2% and 44.5%, respectively, while for healthcare-associated infections, carbapenems were used (23.9%). By AWaRe group, the watch group was predominant for all types (63.9%), for prophylaxis it was the access group (39.3%), and for HAIs it was the reserve group (4.9%).