Among the microorganisms present in the microbiome of Camellia japonica flowers, extracellular vesicles (EVs) derived from Leuconostoc mesenteroides were isolated to investigate their modulatory effects on moisturizing and barrier-related molecular markers. To identify the function of major proteins in L. mesenteroides DB-21-derived extracellular vesicles (LEVs), Gene Ontology (GO) analysis was performed, revealing ATP binding, ribosomal structural proteins, and metal ion binding as predominant molecular-function categories. These proteomic characteristics provide a molecular context that supports the interpretation of the moisturizing and barrier-related responses observed in this study. To further verify new findings, we performed functional evaluations using in vitro and 3D skin models. LEVs increased the mRNA expression level of HAS3, which encodes hyaluronic acid synthase. In addition, the expression levels of filaggrin and involucrin, key proteins involved in skin barrier formation, increased, and these markers were determined a concentration-dependent increase in a 3D artificial skin model. Also, we confirmed that the expression levels of filaggrin and involucrin, which were reduced by UVB damage, were restored when LEVs were applied. In conclusion, LEVs are effective in enhancing various molecular markers related to the skin barrier function, and these results reveal that they hold promise as next-generation microbiome-based functional ingredients.