Background: Children are most susceptible to atopic dermatitis (AD), a chronic, recurrent inflammatory skin condition. Aim: The goal of the current study was to ascertain the frequency of biofilm formation and the expression of the icaA and icaD genes in Staphylococcus aureus (Staph. aureus) strains from AD children emphasizing their relationship with disease severity and antimicrobial resistance. Methods: We conducted a cross-section study on 62 pediatric AD patients. Staph. aureus, which were isolated from the skin and exposed to the microtiter plate method for biofilm formation detection. To find the icaA and icaD genes, the PCR technique was used. Results: The mean age was 6.4±2.7 years. Disease severity was moderate in 58.1% and severe in 25.8% of patients. Positive bacterial cultures were obtained in 88.7% of patients, with Staph. aureus as the most common isolated strains (64.5%). Among S. aureus isolates, 85% produced biofilm and 85% expressed icaA and icaD genes. Severe AD was significantly associated with strong biofilm formation and icaA and icaD genes positivity. Biofilm production was correlated strongly with icaA and icaD expression (P = 0.001), and a significant proportion (75%) of isolates were identified as MRSA. No significant association was found between biofilm production and antibiotic resistance. Conclusion: Pediatric AD patients showed high Staph. aureus colonization, with most strains producing biofilm and carrying icaA and icaD genes, both linked to severe disease. A high proportion were Methicillin-Resistant Staphylococcus aureus (MRSA), posing therapeutic challenges. These findings underscore the necessity of susceptibility-guided therapy and adjunctive antibiofilm strategies.