The in vitro and in vivo antifungal effects of essential oil (EO) extracted by CO2 supercritical fluid extracted from Inula salsoloides flowers on fruits and vegetables were investigated in this study. The results showed that EO had significant activities against all tested fungi in vitro with diameter of inhibition zone greater than 20 mm. The fungus showed extremely high sensitivity to EO and the minimum inhibitory concentration (MIC) was 3.13 mg/mL. The antifungal activities of EO in vivo were investigated by soaking method and fumigation method.. EO showed obvious antifungal activity on grapes with the treatment of EO fumigation method, which significantly slowed the spoilage process of fresh grapes and kept fresh during storage with 0.5 mL EO in vivo. There were 21 EO compounds identified by gas chromatography-mass spectrometry (GC/MS) from Inula salsoloides flowers, of which the content of vanillin was the highest(30.35%) and the antifungal activity of EO was closely related to this composition., The results provide a scientific basis for the development and application of EO of Inula salsoloides flowers in the local food industry.