Fusarium oxysporum（F. oxysporum）is a pathogen that causes Fusarium wilt, root rot, and stem rot in many crops. In this study, the bactericidal effects and mechanism of atmospheric pressure plasma jet (APPJ) on F. oxysporum were studied. Firstly, the effects of processing time, power, and jet distance on the sterilization efficiency were investigated, and the optimal sterilization process and conditions were determined. Secondly, the bactericidal mechanism of APPJ on F. oxysporumwas studied by analyzing the effects of APPJ on the electrochemical properties and active substance content in spore suspension and the effects of APPJ on the mycelial growth, spore morphology and cell membrane integrity of F. oxysporum. The results showed that the optimal conditions for the inactivation of F. oxysporum by APPJ were as follows: power 350 W, treatment time 180 s, jet distance 10.4 cm, the sterilization rate reached a maximum of 99.75%. After APPJ treatment, the colony diameter, mycelial biomass and ergosterol concentration decreased, while the relative conductivity, protein and nucleic acid leakage, propidium iodide (PI) staining intensity, and malondialdehyde (MDA) concentration of F. oxysporumincreased. Scanning electron microscopy and transmission electron microscopy revealed that the surface of F. oxysporum spores, the cell wall membrane, and the interior were wrinkled, destroyed, and vacuolated by APPJ treatment, respectively. Therefore, we concluded that the inactivation of F. oxysporumby APPJ is due to the damaging of the morphology, membrane integrity, and internal structure of spores induced by APPJ.