Defensins are cysteine-rich cationic antimicrobial peptides contributing to the innate immunity in plants. A unique gene encoding a highly cationic bi-domain defensin MtDef5 has been identified in a model legume Medicago truncatula. MtDef5 consists of two defensin domains of 50 amino acids each linked by a 7-amino acid peptide. It exhibits broad-spectrum antifungal activity against filamentous fungi at submicromolar concentrations. It rapidly permeabilizes the plasma membrane of the ascomycete fungi Fusarium graminearum and Neurospora crassa and induces accumulation of reactive oxygen species. It is internalized by these fungi, but uses spatially distinct modes of entry into these fungi. It co-localizes with cellular membranes, travels to nucleus and becomes dispersed in other subcellular locations. It binds to several membrane-resident phospholipids with preference for phosphatidylinositol monophosphates and forms oligomers. Mutations of the cationic amino acids present in the two γ-core motifs of this defensin that eliminate oligomerization also knockout its ability to induce membrane permeabilization and fungal growth arrest. MtDef5 is the first bi-domain plant defensin that exhibits potent broad-spectrum antifungal activity, recruits multiple membrane phospholipids and forms oligomers in their presence. These findings raise the possibility that MtDef5 might be useful as a novel antifungal agent in transgenic crops.