Gray mold caused by Botrytis cinerea is the most harmful postharvest disease responsible for the degradation of strawberries. The presented study targeted the preparation of 7S globulin from pea seeds to serve as an antifungal agent against B. cinerea in vitro and on the post-harvest strawberry to extend the fruits' shelf life. The 7S globulin isolation from pea seeds and characterization employed various methods, such as SDS-PAGE, FTIR, and pH solubility curve. The molecular technique also helped confirm the identity of the causative microorganism of the gray mold disease in strawberries. Utilizing rRNA gene sequencing identified a fungal pathogen that causes gray mold as B. cinerea. The 7S globulin showed three protein bands corresponding to α/ (83 KDa), α (68 KDa), and β (60 KDa) subunits. The isoelectric point was notable at pH 5.8. The essential and non-essential amino acids occurred around 24.92% and 54.04%, respectively. The 7S globulin inhibited the mycelial growth of B. cinerea in a concentration-dependent manner. The Scanning Electron Microscope (SEM) of B. cinerea subjected to 7S globulin showed swelling of both the fungal hyphae and conidia, significantly affected by the pea 7S-globulin treatment, entirely destabilizing and deforming their shape at 0.4 g/L. The 7S-globulin exposure maintained the fruit quality and stopped the strawberry’s natural deterioration. Results further authenticated that 7S globulin (isolated from pea seeds) revealed effective antifungal action against B. cinerea mycelial development via a membrane-targeted mechanism. The 7S globulin affects hyphal morphology, compromises plasma membrane integrity, and prevents post-harvest gray mold on strawberry fruits.