Background: Methicillin Resistant Staphylococcus aureus (MRSA) is a serious pathogen in hospitals leading to failure of treatment. MRSA colonization in pediatric patients increases the risk of severe invasive infections, while in the hospital and once they leave. Objectives: We aimed to study the molecular characters of MRSA and its prevalence among pediatric patients and healthcare workers (HCWs) in National Liver Institute (NLI), also the risk factors and mupirocin efficiency in MRSA decolonization were evaluated. Methodology: Nasal and hand swab specimens were obtained from patients and HCWs in pediatric department of NLI. MRSA detection was achieved by phenotypic methods such as cefoxitin diffusion test, VITEK2 compact system and chromogenic media, then confirmed by PCR. Susceptibility of S. aureus to different antibiotics was done by VITEK2 compact system. All isolated MRSA were subjected to SCCmec typing by multiplex PCR. Results: MRSA prevalence among HCWs was 18.9%, and among ward patients, PICU and NICU was 24.2 %, 16.7 % and 36.4 % respectively. Skin infections and less hygiene training were significant risk factors for colonization with MRSA among HCWs. Regarding antibiogram, high resistance rate was detected among MRSA isolates. Sensitivity and specificity of cefoxitin disc diffusion and VITEK2 were 97.8% and 93.3%, while that of CHROM agar was 91.3% and 80%. According to SCCmec typing, the most predominant SCCmec genotypes in the studied subjects was type III followed by type I then type IV, and the least prevalent was type V in both patients and HCWs. So, hospital acquired MRSA (36 /46) was more than community acquired MRSA strains (10/46 samples). When apply intranasal mupirocin ointment 2% regimen, a high successful decolonization rate was achieved. Conclusion: The most predominant type of SCCmec gene was type III followed by type I, which is linked to hospital acquired strains. Implementation of urgent antibiotic policy and strict infection control programs, in addition to mupirocin decolonization measures will decrease the transmission and spread of these multi drug resistant pathogens.