Fusarium avenaceum (Fr.) Sacc. is an important phytopathogen. Fight against F. avenaceum includes primarily fungicide usage. However, novel strategies are needed in a struggle with F. avenaceum. In this study, terpinolene was used against F. avenaceum as an antimicrobial agent, and the harmlessness of terpinolene was tested on two contrast barley genotypes, Hordeum vulgare L. cv. Cervoise and H. vulgare L. cv. Premium. Firstly, minimum inhibitory concentration (MIC) and half inhibitory concentration (IC50) were detected as 6 and 3 μg μL-1. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was informative about the effects of terpinolene on enniatin and zearalenone biosynthesis in F. avenaceum. Terpinolene leads to the potential decreased enniatin and zearalenone biosynthesis in F. avenaceum. However, no significant differences were recorded for gene expression of aurofusariun biosynthesis. In barley, electroconductivity (EC), catalase (CAT) activity, coupled restriction enzyme digestion-random amplification (CRED-RA), and qRT-PCR assays were tested. No significant ion leakage differences (p>0.05) were detected. Similarly, CAT activity and water loss rate (WLR) values in barley sets were not affected (p>0.05) by terpinolene treatment in majority of experiment sets. Relatively low levels of genomic template instability (75-85%) and epigenetics changes (11-20.6%) were recorded in barley due to terpinolene treatment. WRKY6, WRKY24, and WRKY41 expressions were not significantly changed. The findings showed that terpinolene could be accepted as a potential plant protective agent against phytopathogenic fungi.