Introduction: The emergence of multidrug-resistant (MDR) organisms due to inappropriate antimicrobial use has become a global concern, prompting the implementation of Antimicrobial Stewardship Programs (ASP) in healthcare settings. This study aims to evaluate the impact of ASP on antimicrobial utilization, expenditure, and resistance trends, providing insights into the program’s effectiveness. Methods: An Ambi-directional cohort study was conducted to assess pre- and post-ASP antimicrobial utilization, resistance patterns, and cost trends over one year of ASP- implemented in Mahkota Medical Centre (MMC). A Defined Daily Dose (DDD) per 1,000 patient days was used to quantify antimicrobial consumption, and microbiological resistance data were analyzed. Statistical tests, including the Kruskal-Walli’s test and Student’s t-test, were applied to compare pre- and post-ASP outcomes. Additionally, expenditure trends and cost analysis were assessed to determine the financial impact of the ASP. Results: The mean DDD per 1,000 patient days for ASP-targeted antibiotics was significantly decreased by 17% from 161.52 DDD per 1,000 patient days in the pre-ASP period to 134.49 DDD per 1,000 patient days in the post-ASP period mainly from the usage of third generation cephalosporin, carbapenem and colistin. The annual expenditure for ASP-targeted antibiotics had a significant monetary reduction from RM 30,580.50 in the pre-ASP period to RM 20,590.60 during the post-ASP period. Significant reductions in the mean MDR bacterial resistant rate were notable for extended-spectrum beta-lactamase Escherichia coli (E. coli) (27.48%–17.85%), methicillin-resistant Staphylococcus aureus (22.25%–15.73%) and MDR Acinetobacter spp. (71.46%–49.34%). Conclusion: The implementation of ASP leads to a significant reduction in the ASP-targeted antibiotics utilisation, antimicrobial cost, and MDR bacterial resistance rate. These outcomes are beneficial in the justification and expansion of ASP activities in Malaysia. Keywords: Antimicrobial stewardship; Antibiotic use; Defined Daily Dose; Antibiotic cost; Resistance rate.