Different peptides have been isolated from a wide range of animal species. It is has become increasingly clear that due to the development of antibiotic-resistant microbes, antibacterial and antifungal peptides have attracted the attention in recent years, in order to find new therapeutic agents. In this work, a novel peptide with high inhibitory activity against fungi growth have been isolated from the venom of the Brazilian snake Bothrops jararaca. A Sephacryl S-100 gel filtration column was employed for further separation of proteins. The FV fraction with high antifungal activity was named Pep5Bj, pooled and submitted to reverse-phase chromatography in HPLC. The fraction containing the isolated peptide inhibited the growth of different phytopathogenic fungi (Fusarium oxysporum and Colletotrichum lindemuthianum) and yeast (Candida albicans and Saccharomyces cerevisiae). The peptide minimal inhibitory concentration is comparable to other known antifungal peptides, like insect defensins and cecropins, found in the last years in a large diversity of animals. We investigate F. oxysporum cells membrane permeabilization using SYTOX Green uptake, an organic compound that fluoresces upon interaction with nucleic acids after penetration in cell with compromised plasma membranes. When viewed under fluorescence optical microscopy, F. oxysporum cells exposed to Pep5Bj display strong SYTOX Green fluorescence in the cytosol, especially in the nuclei. The SYTOX Green data suggested that this effect is related to membrane permeabilization. The molecular masses of this peptide was obtained by MALDI-TOF spectrometry and corresponded to 1370Da.