Background: Antibiotic overuse in hospitals is common and linked to adverse outcomes and antimicrobial resistance. Antimicrobial stewardship programs (ASP) aim to optimize prescribing and require context-specific adaptation. Objectives: To describe the experience of implementing and strengthening ASP in hospitals from six Latin American countries by using an integrated framework. Methods: The intervention included a point-prevalence survey (PPS) of antibiotic use, a baseline checklist, a continuous online education program, and individual facility meetings to share SWOT analyses and recommendations. The latter was performed based on PPS and checklist results. The checklist covers six domains (authorities’ commitment, organization, structure, and accountability; interventions; education and training; monitoring and surveillance; and internal communication). The training program spanned 12–18 months and addressed core ASP components. Results: The PPS across 67 hospitals showed an antibiotic use prevalence of 47.9%, with 63% of prescriptions deemed appropriate. The median checklist score was 61.2%. Among the categories assessed, monitoring and surveillance achieved the highest score (median 75.0; IQR 63.9–84.0), while education received the lowest (median 43.8; IQR 29.7–62.5). A total of 80 country groups and 35 individual hospital meetings were held. Conclusions: An integrated, data-driven framework combining PPS, checklists, individual hospital meetings, and sustained training provides a scalable approach to strengthening ASP in diverse Latin American hospitals, aligning with Pan American Health Organization (PAHO) guidance and global recommendations.