Four compounds including a flavone, an acetylenic lactone, a prenylated coumarin, and a 3-methyl ether flavone were isolated from the dichloromethane leaf extract of Baccharis pedunculata (Mill.) Cabr. (Asteraceae). The latter three compounds were identified to be responsible for the antifungal activity against some human pathogenic and phytopathogenic fungi. The most active compound, lachnophyllum lactone, an acetylenic lactone, showed a very high toxicity (LD50 2 micrograms/ml) against human keratinocytes.