Antimicrobial misuse is a worldwide issue, and antimicrobial resistance is considered the most challenging aspect of health care. It has been reported that as much as 30–50% of antimicrobials prescribed in hospitals are deemed unnecessary or inappropriate. Antibiotic stewardship programs (ASPs) include policies that apply continuous management of judicious anti-infectious treatment in the clinical setting. Therefore, the objectives of this study were to evaluate the effect of ASPs on antibiotic consumption, the costs of antibiotic expenditure, and the sensitivity of antimicrobials. A retrospective, quasi-experimental study was performed to assess the effect of ASP at An-Najah National University Hospital, a tertiary care hospital in the West Bank, Palestine, over a period of 20 months before and 17 months after the implementation of the ASP. Data on antibiotic consumption were reported monthly as days of therapy per 1000 patient-days and monthly costs (USD/1000 patient-days). A total of 2367 patients who received one or more of the targeted antibiotics (meropenem, colistin and tigecycline) during their hospital stay were included in the study. They have split into two groups: 1710 patients in the pre-ASP group, and 657 patients in the post ASP group. The most significant reduction in DOT per 1000 patient-days was seen with tigecycline, with a percentage of change of − 62.08%. Furthermore, the mean cost of the three antibiotics decreased significantly by 55.5% in the post-ASP phase compared to the pre-ASP phase. After the implementation of ASP, there was a statistically significant increase in susceptibility to meropenem, piperacillin and piperacillin/tazobactam with respect to Pseudomonas aeruginosa. However, changes in mortality rates were not statistically significant (p = 0.057). ASP positively reduced costs and antimicrobial consumption, with no statistically significant effect on the overall mortality rate. However, a long-term evaluation of the ASP's impact is needed to conclude its lasting impact on infection-related mortality and antimicrobial susceptibility pattern.