The present study was undertaken to find out the antifungal constituents of ethyl acetate soluble fraction of methanolic leaf extract of London rocket weed (Sisymbrium irio L.). To achieve this objective, methanolic leaf extract was partitioned using various organic solvents including ethyl acetate. Different concentrations of ethyl acetate fraction were prepared which ranged from 1.562 to 200 mg mL^‑1, and evaluated against Fusarium oxysporum f. sp. cepae isolated from diseased onion. A significant reduction in fungal biomass of 77–93% was recorded due to different concentrations of this fraction. GC-MS analysis revealed the presence of 7 constituents in the fraction. Among these 1,3-Cyclopentadiene, 5-(1-methylethyllidene) (26.31%) and Di-n-octyl phthalate (26.28%) were the most abundant followed by Acetic Acid, butyl ester (11.67%) and γ-Sitosterol (11.64%). Presence of these compounds may be responsible for antifungal behavior of this fraction against the pathogen of basal rot disease of onion.