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Investigation of changes in the skin microbiome following treatment of atopic dermatitis (AD) with dupilumab may provide valuable insights into the skin microbiome as a therapeutic target. The aim of this study is to assess changes in the AD skin microbiome following treatment of AD with dupilumab (<i>n</i> = 27). E-swabs were collected from nose, lesional, and nonlesional skin before and after 16 weeks of dupilumab therapy, and the microbiome was analyzed by 16S rRNA and <i>tuf</i> gene sequencing. Data for 17 patients with milder disease receiving treatment with non-targeted therapies are also presented. The results show that both groups experienced clinical improvement (<i>p</i> < 0.001) following dupilumab therapy and that Shannon diversity increased and bacterial community structure changed. The relative abundance of the genus <i>Staphylococcus</i> (S.) and <i>S. aureus</i> decreased, while that of S. <i>epidermidis</i> and <i>S. hominis</i> increased. No significant changes were observed for patients receiving non-targeted treatments. The increases in <i>S. epidermidis</i> and <i>S. hominis</i> and the decrease in <i>S. aureus</i> correlated with clinical improvement. Furthermore, changes in <i>S. hominis</i> and <i>S. epidermidis</i> correlated inversely with <i>S. aureus</i>. In conclusion, treatment with dupilumab significantly changed the skin microbiome and decreased <i>S. aureus</i>. Our results suggest a favorable role of commensal staphylococci in AD.