The goal of this study was to evaluate the frequency and role of Staphylococcus aureus infection in patients with atopic dermatitis (AD). In 81 children, ages 2 months to 9 years, affected with moderate to severe AD, 308 samples from the cutaneous lesions were obtained and analyzed. S. aureus was isolated in 52 children (64.2%). Five of these were also colonized by Streptococcus pyogenes and one by Candida albicans. In 61 patients, total IgE serum level and specific IgE were tested to evaluate their allergic status: in 43 children a diagnosis of extrinsic AD was made, while 18 were affected by intrinsic AD. A higher presence of the bacterium was observed in allergic (71%) versus nonallergic children (49%). Our data demonstrate the importance of S. aureus in the clinical manifestation of AD and, in particular, its role in worsening the eczematous lesions of the face, neck, and perineum in children less than 1 year of age.