The antibiotic stewardship programme (ASP) is a new concept initiated by WHO, but nurses are not yet ready to adopt the program. The training and empowerment of nurses are the best strategies for enhancing their knowledge and engagement in ASP. This mixed-method study was used to assess perceived roles and barriers of nurses’ involvement in ASP. An online survey was conducted among 420 clinical nurses to identify their role, and 23 individual interviews were performed among nurses and infection control practitioners to explore the barriers and recommendations to overcome the identified barriers. The majority of the nurses agreed with the sixteen identified roles in ASP, of which ‘antibiotic dosing and de-escalation’ (82.61%), ‘IV to PO conversion of antibiotic, outpatient antibiotic therapy’ (85.23%), and ‘outpatient management, long-term care, readmission’ of the patients (81.19%) had the lowest agreement from the participants. The major themes generated through the qualitative interviews were a lack of knowledge about ASP, poor communication between multidisciplinary teams, lack of opportunity and multidisciplinary engagement, lack of formal education and training about ASP, lack of ASP competency and defined roles in policy, role conflict or power/position, availability of resources, and lack of protected time. Nurses play an integral role in the successful implementation of antibiotic stewardship programs. The empowerment of nurses will help them to adopt the unique role in ASP. Nurses can significantly contribute to antibiotic stewardship efforts and improve patient outcomes through addressing these challenges.