Origanum vulgare subsp. virens (Hoffmanns. & Link) Bonnier & Layens and its essential oil (EO) are widely used in the treatment of respiratory and cutaneous infections in traditional medicine. In order to establish a basis for its traditional use, the antimicrobial activity of the EO of O. vulgare subsp. virens was evaluated against human fungal pathogens. Different oil samples were studied in order to elucidate the intraspecific chemical variability and its impact on the biological activity. Flowering aerial parts of three samples of O. vulgare subsp. virens were collected in different geographical locations and EOs were isolated from air-dried plant material by hydrodistillation. The oils were analysed by GC and GC-MS. Minimal inhibitory and minimal lethal concentrations were measured by broth macrodilution methods for the oils and their main constituents against human pathogenic fungi and the influence of the oils on the filamentation in Candida albicans was assayed. The effect of the oil samples on cell metabolism and cell membrane integrity was studied by flow cytometry. Significant quantitative differences in chemical composition were found between the EO samples and, while the three samples generally displayed potent fungicidal activity, their antifungal potencies varied and appeared to be intensified by increasing carvacrol content. The inhibition of filamentation, on the other hand, may correlate more with γ-terpinene content. The flow cytometry results confirmed the occurrence of damage to the plasma membrane, although not necessarily as a direct effect of the oil on the membrane. The EO of O. vulgare subsp. virens is a broad-spectrum fungicide, thus justifying its potential for use in the treatment of superficial or mucosal fungal infections. The EO shows significant variability in chemical composition between samples, which, in turn, affects its biological activity.