BACKGROUND: High antibiotics use in pediatric intensive care units (PICUs) results in antibiotic resistance, the unfavorable clinical outcome of patients, increase the length of hospital stay, and drug expenditure. AIM: This study aimed at setting clinical guidelines customized according to local diseases epidemiology and local cumulative antimicrobial susceptibility, implementing, and evaluating the Antimicrobial Stewardship Program (ASP) effect in; optimizing antibiotics use, decreasing antibiotics expenditure, decreasing the length of therapy and stay in hospitals, and improving patients’ clinical outcomes. METHODS: A prospective study was conducted at a PICU of the Specialized Pediatric Hospital, Cairo University. Facility-specific guidelines were set, and the ASP was implemented and evaluated through the following indicators; adherence of physicians to the guidelines, ASP recommendations and acceptance of them, the rate of mortality, length of stay, drug costs, antibiotics days of therapy, and length of therapy. RESULTS: The adherence to the ASP guidelines was positively correlated to the patient’s clinical outcome (p = 0.018). In post ASP period, the average length of stay and the length of therapy significantly decreased (p = 0.047, p = 0.001, respectively), the rate of adherence to the ASP guidelines was (91.9%), the days of therapy of ceftazidime, ceftriaxone, and amikacin decreased significantly (p = 0.041, p = 0.026, p = 0.004, respectively). The most common ASP recommendation was drug schedule/frequency change (26.1%) followed by drug discontinuation (17.8%) and the most common antibiotic required intervention was ampicillin-sulbactam (21.6%). CONCLUSION: The antimicrobial stewardship is very effective in optimizing antibiotics use and leads to favorable outcomes in terms of decreased length of therapy, hospital stay, and mortality rate of the patients.