Objective: The engagement with all healthcare professionals involved in antimicrobial use is the key to success for antimicrobial stewardship (AMS) programs. Assessing the awareness and perception of AMS among healthcare professionals is needed to guide the necessary steps required in AMS education. This study primarily aimed to assess awareness and perception of AMS among doctors and nurses across various disciplines in the Universiti Kebangsaan Malaysia Medical Centre (UKMMC). Methods: A cross-sectional study was conducted using a self-administered questionnaire in UKMMC by convenient sampling. The questionnaire consisted of a total of 18 items on demographic data, awareness, perception, and importance on AMS. Results: There were a total of 253 respondents (74 doctors and 179 nurses) with a 74% response rate among doctors and 89.5% among nurses. Doctors (94.6%) were significantly more familiar with the term ‘AMS’ compared to nurses (28.5%) (p < 0.001). Most respondents did not aware of the existence of the AMS program (59.5%) in UKMMC. Institutionalized evidence-based practice guidelines that incorporate local resistance patterns were perceived by the prescribers as the most beneficial (94.6%) and the most-practiced (82.4%) AMS strategy. Doctors (97.3%) were significantly more likely to acknowledge their role in implementing AMS compared to the nurses (52.5%) (p < 0.001). They (97.3%) were also significantly aware of the importance of AMS compared to nurses (70.3%) (p < 0.001). Conclusion: This study highlighted the low awareness of AMS among nurses in a tertiary teaching hospital, which prompts the need for AMS training for this group of healthcare professionals.