Specific mechanisms contributing to the antifungal activity of alpha-thujone, one of the main compounds of essential oils in herbs, against Fusarium graminearum, which causes various diseases of cereals, were studied for the first time. Minimum inhibitory concentration (MIC) value of alpha-thujone was 3.2 mu g mu L-1 for F. graminearum. Its toxigenic effect was confirmed by WST-1-based cytotoxicity assay. Due to alpha-thujone treatment, changes in genomic template stability (GTS), type II and III methylation profiles were detected. GTS rates were calculated as 89.33 and 94% according to random amplified polymorphic DNA (RAPD) and inter-retrotransposon amplified polymorphism (IRAP) assays, respectively. Mean values of polymorphisms were 28.92 and 36.42% by evaluating digestion profiles. Gene expression analysis showed alpha-thujone caused upregulation of mgv1, mst20 and cat genes but downregulation of tri5. Fold change values were 1.86 +/- 0.14, 3.10 +/- 0.03, 2.56 +/- 0.078 and 0.43 +/- 0.15 respectively. The acridine orange-ethidium bromide (Ao/Eb) and 2 ',7 '-dichlorodihydrofluorescein diacetate (DCF-DA) staining assays revealed that alpha-thujone treatment triggers apoptosis and oxidative stress in F. graminearum at cellular level. Additionally, 6.51-fold increase in catalase activity in comparison with the control was detected. There were no statistically significant changes in water loss rates (WLRs) when Hordeum vulgare L. cv. Akhisar seedlings were treated with alpha-thujone. According to the water loss rate values, alpha-thujone is not a potential abiotic stress factor that negatively affects the physiological development of barley plantlets. Outcomes showed that alpha-thujone leads to serious morphological, genetic, epigenetic, cellular alterations on F. graminearum, so could be a candidate as an alternative herbal antifungal compound.