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Introduction Antibiotic resistance is a well-known public health problem. Many studies indicate that there is an overuse in the hospital setting, and that is responsible for the threat of bacterial resistances. Pharmacists play an important role in implementation of guidelines in hospital, and their proactive attitude related to antibiotic use and bacterial resistances could improve antibiotic use in hospitals. This study sought to identify in what situations pharmacists agree that they should reject the supply of antibiotics after medical prescription to hospitalized patients. Method We conducted a cross-sectional study covering all hospital pharmacists registered with the Official College of Pharmacists in Portugal's Central Region. A self-administered questionnaire with questions about situations that the supply of antibiotics by pharmacists should be refused in the hospital setting was mailed to hospital pharmacists, whose responses ranged from 0 (total disagreement) to 10 (total agreement). Results About 50% of all hospital pharmacists participated in this survey. The highest agreement to reject the supply of antibiotics was identified for situations where prescribed dosage is not appropriate and when the pharmacist believes that the prescribed antibiotic is not the most appropriate for the clinical situation. About half of the participants did not show full agreement with the rejection of antibiotics when drug interactions are identified. Conclusion Interaction between pharmacists and physicians is very important to improve antibiotic use in hospital settings.