Antimicrobial peptides play a pivotal role in the innate immunity of plants. Defensins are cysteine-rich antifungal peptides with multiple modes of action. A novel Oleaceae-specific defensin gene family has been discovered in the genome sequences of the wild and cultivated species of a perennial olive tree， Olea europaea. OefDef1.1， a member of this defensin family， potently inhibits the growth of ascomycete fungal pathogens Botrytis cinerea and three Fusarium spp. in vitro. OefDef1.1 rapidly permeabilizes the plasma membrane of the conidial and germling cells of B. cinerea. Interestingly， it induces reactive oxygen species and translocates to the cytoplasm only in the germlings， but not in the conidia. In medium containing high concentration of Na1+， antifungal activity of OefDef1.1 is significantly reduced. Surprisingly， a chimeric OefDef1.1 peptide containing the γ-core motif of a Medicago truncatula defensin MtDef4 displays Na1+-tolerant antifungal activity. In a phospholipid-protein overlay assay， the chimeric peptide exhibits stronger binding to its phosphoinositide partners than OefDef1.1 and is also more potent in inhibiting the gray mold disease on the surface of Nicotiana benthamiana and lettuce leaves than OefDef1.1. Significant differences are observed among the four ascomycete pathogens in their responses to OefDef1.1 in growth medium with or without the elevated concentration of Na1+. The varied responses of closely related ascomycete pathogens to this defensin have implications for engineering disease resistance in plants.