Fusarium oxysporum f. sp. pisi (van Hall) Snyder & Hansen is an important pathogen of pea that causes wilt. The present study was carried out to evaluate the efficacy of rhamnolipid biosurfactant produced by newly isolated Pseudomonas aeruginosa strain SS14 as an antifungal agent against F. oxysporum f. sp. pisi in Pisum sativum L. The bacterial strain P. aeruginosa SS14 was isolated from crude oil contaminated soil and identified by 16S rDNA sequencing. The biosurfactant was characterized as rhamnolipid by FTIR and LC–MS analyses. Treatment of pea seeds and seedlings under natural conditions of light, temperature and humidity with the rhamnolipid at a concentration of 25 µg ml−1 prior to sowing or planting in pathogen laden soil resulted in complete suppression of characteristic wilt symptoms. The results demonstrate the possibility to develop a sustainable and eco-friendly control measure against F. oxysporum f. sp. pisi which is currently not available.