Fusarium head blight (FHB), caused by Fusarium graminearum, is a predominant disease of wheat. Due to the lack of disease-resistant germplasm, chemical control is an important means to control wheat scab. Volatile substances produced in near-isogenic wheat lines were detected after inoculation with F. graminearum, and 4-propylphenol, which appears in FHB-resistant lines, was identified. In vitro and in vivo antifungal activity tests demonstrate that 4-propylphenol effectively inhibits the mycelial growth of F. graminearum. Metabolomics analysis showed changes in glutathione metabolism, indicating that 4-propylphenol triggered reactive oxygen species (ROS) stress. This was consistent with the increasing ROS levels in Fusarium cells treated with 4-propylphenol. Further results demonstrated that excessive accumulation of ROS induced DNA and cell membrane damage in the mycelium. Moreover, 4-propylphenol showed different degrees of inhibition against other soil-borne pathogens (fungi and oomycetes). These findings illustrated that 4-propylphenol has broad spectrum and high antifungal activity and should be considered for use as an ecological fungicide.