<p>Recent research has underscored the significant role of skin fungi in human health and disease. The advent of next-generation sequencing has facilitated the identification of previously unrecognized fungi; however, a standardized sampling method for the skin mycobiome has yet to be established. Given that fungi are distributed across all layers of the skin, this study aimed to establish and compare the fungal profiles obtained from paired skin swabs (skin surface) and tissue specimens (skin dermis), utilizing ITS rRNA gene sequencing. Fungal diversity and composition varied between swabs and tissue specimens from the same individual. Tissue specimens exhibited greater fungal diversity relative to skin swabs, as indicated by the Chao1 estimator (<i>P</i>=0.028) and Shannon index (<i>P</i>=0.0136). Notably, <i>Malassezia</i> species was more abundant in swabs (<i>P</i>=0.002), whereas <i>Trichoderma</i> species was more prevalent in tissue samples (<i>P=</i>0.0001). Compared to swabs, tissue sampling also demonstrated superior sensitivity in detecting non-<i>Malassezia</i> species. These findings suggest that skin swabs and tissue specimens provide different views of the skin mycobiome: Swabs are effective for studies targeting surface-dwelling fungi, while tissue sampling remains necessary when investigating deeper skin fungi and potential systemic infections. Future research should carefully consider appropriate sampling method based on target fungal location to mitigate procedural artifacts.</p><p>Level of Evidence III This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors <a href="http://www.springer.com/00266">www.springer.com/00266</a>.</p>