In recent years, studies have demonstrated the function of many antimicrobial peptides against an extensive number of microorganisms that have been isolated from different plant species and that have been used as models for the study of various cellular processes linked to these peptides' activities. Recently, a new defensin from Phaseolus vulgaris (L.) seeds, named PvD(1,) was isolated and characterized. PvD(1) was purified through anion exchange and phase-reverse chromatography. PvD(1)'s antifungal activity was tested. A SYTOX Green uptake assay revealed that the defensin PvD(1) is capable of causing membrane permeabilization in the filamentous fungi Fusarium oxysporum, Fusarium solani, and Fusarium laterithium and in yeast strains Candida parapsilosis, Pichia membranifaciens, Candida tropicalis, Candida albicans, Kluyveromyces marxiannus, and Saccharomyces cerevisiae at a concentration of 100 μg/ml. Ultrastructural analysis of C. albicans and C. guilliermondii cells treated with this defensin revealed disorganization of both cytoplasmic content and the plasma membrane. PvD(1) is also able to inhibit glucose-stimulated acidification of the medium by yeast cells and filamentous fungi, as well as to induce the production of reactive oxygen species and nitric oxide in C. albicans and F. oxysporum cells.