| Summary: | This study investigated the potential of <i>Thymus serpyllum</i> L. and <i>Mentha × piperita</i> L. essential oils (EOs), known for their bioactive properties, as adjunctive treatments targeting Basal cell carcinoma cancer stem cells (BCC CSCs). Primary cultures were established from ten BCC tumor samples and their distant resection margins as controls. The chemical composition of the EOs was analyzed by gas chromatography–mass spectroscopy (GC-MS) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The biological effects were evaluated via colony and spheroid formation, scratch assays, MTT and neutral red cytotoxicity assays, and qRT-PCR for Hh (SHH, PTCH1, SMO, and GLI1) and Notch (Notch1 and JAG1) gene expression. GC analysis identified thymol, p-cymene, and linalool as the main components of the EO of <i>T. serpyllum</i> L., and menthone and menthol in the EO of <i>M. × piperita</i> L. IC50 values were 262 µg/mL for <i>T. serpyllum</i> L. and 556 µg/mL for <i>M. × piperita</i> L. and were applied in all experiments. Both EOs significantly reduced CSC clonogenicity and migration (<i>p</i> < 0.05). The EO of <i>T. serpyllum</i> L. downregulated SMO and GLI1, while the EO of <i>M. × piperita</i> L. upregulated PTCH1, Notch1, and JAG1 (<i>p</i> < 0.05). These findings suggest that both EOs exhibit anticancer effects in BCC CSCs by modulating key oncogenic pathways, supporting their potential in BCC therapy.
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