AimsTo understand the mode of action of thyme essential oil as an alternative biofungicide. Methods and ResultsThe chemical composition of thyme essential oil isolated by hydrodistillation from the aerial parts of Thymus vulgaris was analyzed. The main constituents of thyme essential oil were thymol (7696%), -cymene (989%), -terpinene (192%) and caryophyllene oxide (169%). The antifungal activity of the oil and its pure major component (thymol) was assessed by the invitro assay against Mycosphaerella graminicola. Thyme oil exhibited higher antifungal activity than thymol. The expression pattern of genes involved in fungal development and detoxification acting in M. graminicola under thyme oil and thymol treatment was analyzed. Thyme oil overexpressed, more than thymol, the genes encoding for the efflux pump (MgMfs1, MgAtr4), the regulatory subunit of protein kinase A (PKA) (MgBcy1) and the MAPK MgHog1. Thyme oil repressed the expression of the genes encoding for the efflux pump MgAtr4, the MAPK (MgSlt2) and the regulatory subunit of PKA (MgBcy1). However, thymol repressed only MgAtr4 and MgSlt2 expression. ConclusionsThese data highlight the ability of thyme oil to target genes involved in fungal development and virulence of the yeast-like fungi M. graminicola, which explain its higher antifungal activity. Significance and Impact of the StudyThese findings will probably be useful to design an alternative biofungicide which will not lead to pathogen multidrug resistance.