Background: Atopic dermatitis (AD) is a major public health problem worldwide characterized by significant colonization of Staphylococcus aureus (S. aureus) in the skin lesions. This study focused on investigating the association between methicillin susceptibility and biofilm production by S. aureus isolates from AD patients and correlating them with the severity of the disease. Methods: Out of 108 AD patients, sixty- six S. aureus isolates were isolated and subjected for detection of biofilm production by microtiter plate method. PCR was performed to detect mec A, ica A, and ica D genes. Results: Biofilm production among S. aureus isolates was 83.3%. There was a high statistically significant association between the severity of AD and biofilm production (p<0.001). 86.4% of isolates were confirmed as methicillin resistant Staphylococcus aureus (MRSA) by mec A gene detection. There was statistically significant association between methicillin resistance and biofilm production and ica A and D genes expression (p <0.001), as well as the severity of AD and positive ica A and D genes (p=0.003 for each). No significant association was detected between the severe form of AD and mec A gene expression (p=0.07). Conclusion: Severity of AD correlates with S. aureus ability to produce biofilm. Although MRSA was found in a high percentage colonizing AD patient especially in the severe form, it does not affect the severity of the disease.