Background: The inappropriate and excessive use of antibiotics is a global health issue that significantly affects children. One of the main causes of antibiotic misuse, which might contribute to the phenomenon of antibiotic resistance, is mothers' ignorance of the right way to use antibiotics. Aim: This study aimed to evaluate the efficacy of guidelines intervention on mothers' awareness regarding proper antibiotic usage and resistance. Design: A quasi-experimental research design. Setting: This study was carried out in a pediatric outpatient clinic, affiliated to Ain Shams Pediatric Hospital, Cairo Governorate, Egypt. Sampling: convenience sampling composed of 112 mothers who had children aged ≤ 5 years old. Tools: Three tools were utilized in this study: 1) Antibiotic knowledge questionnaire for mothers. 2) Mothers' attitudes regarding antibiotic usage and resistance. 3) Mothers' self-reported practices regarding antibiotics. Results: revealed that 66.1% of mothers had poor knowledge about antibiotic use and resistance pre-intervention, which improved after the intervention to good knowledge (75%, 63.2%) in post-, and follow-up respectively. The minority (8.9%, 13.4%) of mothers had positive attitudes and satisfactory reported practices about antibiotic use and resistance in pre-intervention while increasing to the majority (87.5%, 83%) in the post and to more than three-quarters (79.2%, 76.4%) at follow-up intervention respectively. Conclusion: The guidelines intervention and health education session had a significant impact on improving mothers' knowledge, attitudes, and practices related to antibiotics use and its resistance. Recommendation: The study recommends that healthcare providers should educate mothers to reduce the development of antibiotic resistance and preserve the effectiveness of antibiotics for future generations.