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<i>Fusarium graminearum</i> is a harmful pathogen causing head blight in cereals such as wheat and barley, and thymol has been proven to inhibit the growth of many pathogens. This study aims to explore the fungistatic effect of thymol on <i>F. graminearum</i> and its mechanism. Different concentrations of thymol were used to treat <i>F. graminearum</i>. The results showed that the EC₅₀ concentration of thymol against <i>F. graminearum</i> was 40 μg/mL. Compared with the control group, 40 μg/mL of thymol reduced the production of Deoxynivalenol (DON) and 3-Ac-DON by 70.1% and 78.2%, respectively. Our results indicate that thymol can effectively inhibit the growth and toxin production of <i>F. graminearum</i> and cause an extensive transcriptome response. Transcriptome identified 16,727 non-redundant unigenes and 1653 unigenes that COG did not annotate. The correlation coefficients between samples were all >0.941. When FC was 2.0 times, a total of 3230 differential unigenes were identified, of which 1223 were up-regulated, and 2007 were down-regulated. Through the transcriptome, we confirmed that the expression of many genes involved in <i>F. graminearum</i> growth and synthesis of DON and other secondary metabolites were also changed. The gluconeogenesis/glycolysis pathway may be a potential and important way for thymol to affect the growth of <i>F. graminearum</i> hyphae and the production of DON simultaneously.