MsDef1 and MtDef4 from Medicago spp. are small cysteine-rich defensins with potent antifungal activity against a broad range of filamentous fungi. Each defensin has a hallmark γ-core motif (GXCX3-9 C), which contains major determinants of its antifungal activity. In this study, the antifungal activities of MsDef1, MtDef4, and peptides derived from their γ-core motifs, were characterized during colony initiation in the fungal model, Neurospora crassa. These defensins and their cognate peptides inhibited conidial germination and accompanying cell fusion with different potencies. The inhibitory effects of MsDef1 were strongly mediated by the plasma membrane localized sphingolipid glucosylceramide. Cell fusion was selectively inhibited by the hexapeptide RGFRRR derived from the γ-core motif of MtDef4. Fluorescent labelling of this hexapeptide showed that it strongly bound to the germ tube plasma membrane / cell wall. Using N. crassa expressing the Ca(2+) reporter aequorin, MsDef1, MtDef4 and their cognate peptides were each shown to perturb Ca(2+) homeostasis in specific and distinct ways, and the disruptive effects of MsDef1 on Ca(2+) were mediated by glucosylceramide. Together, our results demonstrate that MsDef1 and MtDef4 differ markedly in their antifungal properties and specific domains within their γ-core motifs play important roles in their different modes of antifungal action.