Seseli L. is an important genus with high number of aromatic species used in traditional medicine due to recognized therapeutic properties of their essential oils, namely antimicrobial activity. However, and in order to explore its potential utilization for industrial and commercial purposes, scientific studies addressing the chemical composition, the action mechanism and the toxicological safety of the oils are missing. The present study was therefore designed to elucidate the chemical and biological effects of the essential oils of Seseli tortuosum and of Seseli montanum subsp. peixotoanum . Given that essential oils are currently used in topical applications we assessed its cytotoxicity in human keratinocytes. The oils were investigated by GC and GC–MS and the antifungal activity (MIC and MLC) was evaluated against yeasts, dermatophyte and Aspergillus strains. Assessment of keratinocytes viability after essential oils treatment was made through the colorimetric assay MTT. It is possible to distinguish the oils of the two taxa through differences in the contents of Z-β-ocimene, limonene and β-elemene. The results also demonstrated that the oil of S. tortuosum has higher anti-fungal activity than S. montanum oil. Furthermore, it was also observed that S. tortuosum essential oil has cytotoxic properties to human cells when used in concentrations higher than 0.64 μL/mL. In conclusion, it is possible to find appropriate doses of S. tortuosum oil showing both antifungal activity and very low detrimental effect in human keratinocytes.