Fusarium oxysporum: f. sp. niveum (Fusarium oxysporum) causes watermelon wilt, drastically reducing its yield. Thymol (5-methyl-2-(1-methylethyl) phenol, PubChem CID: 6989) has been extensively reported to have antimicrobial activity against pathogenic microorganisms. In this study, the growth of F. oxysporum was significantly inhibited by thymol in vitro. The dry weight of F. oxysporum was decreased significantly. Thymol-induced cell membrane damage was observed at 24 hpi (hours post-incubation). Therefore, the changes in the gene expression level of F. oxysporum after treatment with 80 mu g/mL (average EC50 value) of thymol were analyzed using RNA-Seq to reveal the underlying fungicidal mechanisms of thymol. Among the 5057 differentially expressed genes (DEGs), 2440 and 2617 were up-regulated and down-regulated, respectively. Bioinformatics analysis demonstrated that the expression levels of most genes, including glycosphingolipid biosynthesis and sphingolipid metabolism, were down-regulated. However, the genes involved in antioxidant activity, chitin biosynthesis, and cell wall modification were up-regulated. These results were verified by quantitative reverse transcription PCR (qRT-PCR). According to these results, thymol produces reactive oxygen species (ROS) accumulation and destroys the integrity of the cell wall and cell membrane by inhibiting the genes involved in cell wall and cell membrane synthesis.