(-)-Spirobrassinin, a glucosinolate compound from Brassicaceae plants, has shown inhibitory effects on weeds. However, its specific target sites are not well explored. This study used molecular docking, molecular dynamics simulations, and biological experiments to investigate (-)-Spirobrassinin’s target sites. The inhibitory effects of (-)-Spirobrassinin were observed on various enzymes crucial for plant metabolic pathways, including dihydroxyacid dehydrogenase, 4-hydroxyphenylpyruvate dioxygenase, protoporphyrinogen oxidase, and acetolactate synthase. Additionally, it disrupts the metabolism of vital phytohormones, namely abscisic acid and gibberellin. Molecular dynamics simulations revealed stable interactions between (-)-Spirobrassinin and specific residues (Phe270 and Phe261) of the photosystem II D1, involving electrostatic, hydrophobic, and van der Waals forces. This study provides valuable insights into (-)-Spirobrassinin’s mode of action, highlighting its potential as a natural herbicide from Brassicaceae plants.