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Patients with atopic dermatitis (AD) are more likely than healthy individuals to harbour Staphylococcus aureus on their skin. Superantigens (SAgs) produced by specific S. aureus strains may contribute to AD-associated skin inflammation. The present study compared the prevalence and types of SAg-encoding genes between S. aureus isolated from patients with AD and from controls, and within the AD group between isolates from different sampling sites (lesional skin, non-lesional skin, and nares). This retrospective case-control study extracted data from 2 previous studies that examined S. aureus using whole-genome sequencing. The 138 S. aureus isolates obtained from 71 AD patients contained 349 SAg-encoding genes; 22 (6.3%) were found in isolates from nares (0.4 ± 0.6 genes per isolate), 99 (28.4%) in isolates from non-lesional skin (3.7 ± 3.9), and 228 (65.3%) in isolates from lesional skin (4.2 ± 4.5). S. aureus (n = 101) from the control group contained 594 SAg-encoding genes (5.9 ± 4.2). Of the S. aureus isolated from lesional AD skin, 69% carried at least 1 gene encoding SAg compared with 33% of AD nasal isolates. SAg could be a factor in the pathogenesis of a subset of AD patients.