Significance Several nodule-specific cysteine-rich (NCR) peptides expressed in a model legume Medicago truncatula exhibit potent antimicrobial activity. However, their structure–activity relationships and mechanisms of action against fungal pathogens of plants are still largely unknown. A small highly cationic peptide NCR044 with potent antifungal activity has been identified. This peptide has a unique highly dynamic structure and exhibits multifaceted mechanisms of action against a fungal pathogen Botrytis cinerea . Exogenous application of this peptide confers resistance to a gray mold disease caused by B. cinerea in tobacco and tomato plants as well as postharvest products. Our work paves the way for future development of NCR peptides as spray-on antifungal agents.