Terpenoids, one of the major components of essential oils, are known to exert potent antifungal activity against yeast Saccharomyces cerevisiae. They have been the subject of a considerable number of investigations that uncovered extensive pharmacological properties, including antifungal and antibacterial effects. However, their mechanism of action remains elusive. In order to use terpenoids as the antimicrobial and antifungal agents in food preservation in a rational way, a good knowledge of their mode of action is required. We hypothesized that the cellular membrane is the main target site for the antifungal agents, and that structural properties of these agents are key to penetrate and act upon phospholipid bilayers. In this study, we thus aimed to study the effect of terpenoids on the cell membrane integrity, with the focus on both their structural properties, such as the presence of aromatic ring or hydroxyl group; and their hydrophobicity, as a consequence of these structural features. We first uncovered the antifungal properties of phenolic terpenoids thymol, carvacrol and eugenol, cyclic terpenes limonene, carveol, and α-pinene, in addition to the closely related compounds of different chemical structures. We then examined the cell membrane deterioration upon the addition of these reagents. Our results demonstrate that the presence of a phenolic -OH moiety is crucial, and hydrophobicity gained by the aromatic ring structure contributes to the ability of penetration and damaging yeast plasma membrane to achieve high antifungal activity.