Postharvest fungal diseases remain a major cause of fruit losses during storage and distribution. In apples, grey mould caused by Botrytis cinerea is one of the most important postharvest pathogens, leading to significant economic losses worldwide. Although chemical fungicides are widely used to control this disease, increasing concerns about toxicity, environmental impact, and pathogen resistance have stimulated the search for safer and more sustainable alternatives. In this study, plant-derived antimicrobial peptides from the South American native species Peltophorum dubium were evaluated as potential natural antifungal agents for postharvest disease control. Four defensin candidates were selected from a catalogue of antimicrobial peptides previously generated through transcriptome mining of germinated seeds. Antifungal activity against B. cinerea was initially assessed using soluble protein fractions from recombinant production in Escherichia coli. Subsequently, the peptides PdDf11 and PdDf13 were purified from the two most active fractions; both peptides completely inhibited fungal growth in vitro, with a minimum inhibitory concentration of 6 µM. Finally, their protective effect was evaluated in apples stored under cold conditions. Purified PdDf11 showed the highest efficacy, reducing disease severity by 97% and disease incidence by 86%. These results highlight defensins as promising natural antifungal agents for sustainable postharvest protection of apples.