The soil-born filamentous fungal pathogen Fusarium oxysporum f. sp. cubense (FOC), which causes vascular wilt disease in banana plants, is one of the most economically important Fusarium species. Biocontrol using endophytic microorganisms is among the most effective methods for controlling banana Fusarium wilt. In this study, volatile organic compounds (VOCs) showed strong antifungal activity against FOC. Seventeen compounds were identified from the VOCs produced by endophytic fungi Sarocladium brachiariae HND5, and three (2-methoxy-4-vinylphenol, 3,4-dimethoxystyrol and caryophyllene) showed antifungal activity against FOC with 50% effective concentrations of 36, 60 and 2900 μL/L headspace, respectively. Transmission electron microscopy (TEM) and double fluorescence staining revealed that 2-methoxy-4-vinylphenol and 3,4-dimethoxystyrol damaged the plasma membranes, resulting in cell death. 3,4-dimethoxystyrol also could induce expression of chitin synthases genes and altered the cell walls of FOC hyphae. Dichloro-dihydro-fluorescein diacetate staining indicated the caryophyllene induced accumulation of reactive oxygen species (ROS) in FOC hyphae. FOC secondary metabolism also responded to active VOC challenge by producing less fusaric acid and expressions of genes related to fusaric acid production were interrupted at sublethal concentrations. These findings indicate the potential of S. brachiariae HND5 as a biocontrol agent against FOC and the antifungal VOCs as fumigants.