Introduction: Antibiotics are vital for treating bacterial infections, but misuse has contributed to antibiotic resistance, a global health threat. Understanding healthcare professionals' prescribing practices and adherence to guidelines is essential for effective antibiotic stewardship. Objective: This study assesses healthcare professionals' knowledge, attitudes, and practices regarding antibiotic use at Elmiek Nimer University Regional Hospital, identifying barriers to guideline adherence. Methods: A cross-sectional study was conducted using a structured questionnaire among healthcare professionals. It covered demographics, knowledge of guidelines, prescribing practices, participation in stewardship programs, and perceived barriers. Descriptive statistics were used for analysis. Results: Most participants were registrars (41.5%) and medical officers (34.1%), with 35.7% having 5-10 years of experience. Despite moderate to high self-rated antibiotic knowledge (60.2% moderate, 40% high), 78% were unaware of hospital guidelines. 77.2% frequently prescribed antibiotics, guided mainly by clinical symptoms (85.4%) and lab results (69.1%). Only 15.4% had engaged in stewardship programs, finding them effective. Barriers included financial constraints (47.2%) and medication-related issues (46.3%). Conclusion: There are gaps in guideline awareness and adherence, even with moderate knowledge of antibiotics. Enhanced stewardship programs, targeted training, and hospital-specific guidelines are necessary to curb antibiotic resistance. Addressing financial and medication-related barriers is crucial for better adherence. Recommendations: Implement comprehensive stewardship programs with tailored training. Collaborate with policymakers and pharmaceutical providers to alleviate financial and medication barriers. Develop and disseminate hospital-specific guidelines for consistent practice.