UNLABELLED: Vulvar lichen sclerosus (VLS) is a chronic skin condition affecting the vulva, causing significant discomfort, but its etiology in prepubertal girls remains poorly understood. VLS presents with itching, irritation, and pain. Recent studies suggest that cutaneous dysbiosis might play a role in VLS. Our study aims to investigate differences in the vulvar skin microbiota among prepubertal girls with VLS, those with labial adhesions, and healthy controls, and to explore potential microbial links to VLS. We performed a comparative analysis of 16S ribosomal RNA (rRNA) sequences from vulvar skin samples of 18 girls with VLS, 15 girls with labial adhesions, and 11 healthy girls. Microbial diversity was assessed using α diversity, β diversity, and LEfSe, and functional microbial pathways were predicted. No differences were observed in α diversity among groups. However, β diversity analysis revealed significant differences in microbial composition (Jaccard, IMPORTANCE: Cutaneous dysbiosis in vulvar lichen sclerosus (VLS) may play a key role in disease pathogenesis, especially when specific microbial imbalances persist in affected patients. However, most clinical evaluations focus on symptoms rather than microbial composition, risking missed opportunities for microbiome-targeted interventions. Thus, this study highlights the importance of microbiota surveillance as a potential tool for improving the diagnosis and treatment of VLS.