In this study, the antifungal activity of peptides extracted from Adenanthera pavonina seeds was assessed. Peptides were extracted and fractionated by DEAE-Sepharose chromatography. The non-retained D1 fraction efficiently inhibited the growth of the pathogenic fungi. This fraction was later further fractionated by reversed-phase chromatography, resulting in 23 sub-fractions. All separation processes were monitored by tricine-SDS-PAGE. Fractions H11 and H22 strongly inhibited the growth of Saccharomyces cerevisiae and Candida albicans. Fraction H11 caused 100% death in S. cerevisiae in an antimicrobial assay. The complete amino acid sequence of the peptide in fraction P2 was determined, revealing homology to plant defensins, which was named ApDef1. Peptides from fraction H22 were also sequenced.