The antifungal mechanism of potato glycoalkaloids was studied using a sensitive species, Fusarium solani. The effects of potato glycoalkaloid extract on the ultrastructure, membrane permeability, and contents of reducing sugar, soluble sugar, soluble protein, and mycelial fat of F. solani were determined. Potato glycoalkaloids significantly affected F. solani mycelial morphology, resulting in bubbly mycelial cell walls, incomplete outer layer, discontinuous cell membrane, disorganized structures of mitochondria and other organelles, and visible leakage of cell contents. Investigation of material metabolism indicated that potato glycoalkaloids disrupted selective permeability of mycelial cell membranes; caused massive exudation of internal lipids, proteins, and carbohydrates; hindered hydrolysis of reducing sugar; affected nutrient absorption and utilization; and inhibited decomposition metabolism of mycelia. Thus, potato glycoalkaloids altered the morphology of fungal mycelia, destroyed cell membrane structure, increased mycelial cell membrane permeability, and caused cell contents leakage, resulting in effective inhibition of growth and metabolism of plant pathogenic fungi and so could decrease the occurrence of plant disease.