Aims: The aim of this study was to determine the biodiversity of the skin staphylococcal community from patients with atopic dermatitis (AD) and superantigen (SAg) detection from Staphylococcus aureus isolates. Methods and Results: In this study, we developed a novel multiplex PCR that allows the identification and discrimination of bacteria belonging to the Staphylococcus genus both Staph. aureus and coagulase-negative Staphylococcus À Staph. capitis, Staph. epidermidis, Staph. haemolyticus and Staph. hominis isolated from the skin of patients with AD. In addition, a multiplex PCR assay that allows the rapid screening of the 19 genes that encode staphylococcal enterotoxins (SEs), SE-like toxins and toxic shock syndrome toxin-1 was also performed and applied in Staph. aureus isolates. The microflora of the skin of patients with AD was dominated by Staph. aureus (69 isolates, 35Á6%) followed by Staph. epidermidis (59 isolates, 30Á4%) species. The SElM and SElN genes were the most frequently detected in our study (15 isolates, 71Á4%), followed by SEG and SElO (14 isolates, 66Á7%). Conclusions: Our molecular-based approach successfully identified the staphylococcal microflora that was relatively specific to patients with AD. Considering skin colonization and expression of virulence factors, the Staph. aureus may play a relevant role in AD pathophysiology. Significance and Impact of the Study: This ability to classify disease-related microbial species provides new insights into the relevance of those microbes in human disorders.