Background: Chemical fungicides are widely used to control crop diseases, but these chemicals have adverse effects. They destroy the ecological environment and even have toxic effects on human beings. In this context, the development of botanical pesticides is relevant. One potential botanical pesticide is chelerythrine, a main alkaloid of Chelidonium majus L., which has high antitumor, fungistasis, and antiphlogosis bioactivity. Objective: This study was designed to present an ultrasonic extraction method for chelerythrine and spore germination experiments to inhibit pathogenic fungi. Fungistasis of chelerythrine is now centralized in basic microbiology experiments, such as observing bacteriostatic rings. This study investigates chelerythrine based on pathogenic fungal spore germination and the influence of germ tube elongation. Materials and Methods: Samples of C. majus L., which were wild used in this experiment, were picked from Harbin experimental forest farm of Northeast Forestry University. An L9 (34) orthogonal experiment was designed to optimize the ultrasonic extraction method. All the plant pathogenic fungus strains used in the experiment were preservation strains of Northeast Forestry University Microbial preservation center. Pathogenic fungi were cultivated by joining chelerythrine with and observed germ tube growth and spore germination. Results: The optimum ultrasonication extraction process for chelerythrine has a liquid/solid ratio of 1:8, 35 min of extraction time, 85% of ultrasonic frequency, and 75% of ethanol concentration. When the concentration of chelerythrine was 1.7 × 10−2 mg/ml, the inhibition rates of Septoria microspora Speg. spores and Curvularia lunata were 96.67% and 84.94%, respectively. Moreover, when the concentration of chelerythrine was 1.7 × 10−6 mg/ml, the inhibition rates of S. microspora spores and C. lunata were 47.64% and 12.05%, respectively. Conclusion: Fungistasis activity reached a high level with 1.7 × 10−6 mg/ml of chelerythrine. Chelerythrine has the characteristics of less dosage and obvious fungistasis and has a good prospect for botanical fungicide development.