This article studies the sterilization effects of high-voltage pulsed electric field (PEF) of technology on filamentous fungi. A cell dielectric model was proposed based on the physical structure of filamentous fungi. Basic theories of the electromagnetic field were comprehensively applied, and the multiphysics field simulation software COMSOL Multiphysics was used for more detailed study. The effects of PEF treatment parameters and microbial characteristic parameters on the resulting cell membrane and nuclear membrane changes were simulated and analyzed. The results showed significant effects on the transmembrane voltage of the cell membrane and nuclear membrane from the electric field intensity, pulse duration, cell membrane thickness, superposition effect of the pulses. However, the amount of hyphae had little effect, and the number of cell nuclei and the thickness of the cell walls had almost no effect on the transmembrane voltage of the cell membranes and the nuclear membranes. The results provide theoretical support for applying high-voltage PEFs to kill fungi in practical applications.