Phorbol ester extraction was carried out from Jatropha curcas seed cake, a by-product from the biodiesel fuel industry. Four repeated extractions from 5 g of J. curcas seed cake using 15 ml of 90% (v/v) ethanol and a shaking speed of 150 rpm gave the highest yield of phorbol esters. The ethanolic extract of J. curcas seed cake showed antifungal activities against important fungal phytopathogens: Fusarium oxysporum, Pythium aphanidermatum, Lasiodiplodia theobromae, Curvularia lunata, Fusarium semitectum, Colletotrichum capsid, and Colletotrichum gloeosporioides. The extract contained phorbol esters mainly responsible for antifungal activities. The extract could therefore be used as an antifungal agent for agricultural applications.