Antimicrobial peptides are an abundant group of proteinaceous compounds widely produced in the plant kingdom. Among them, the γ-thionin family, also known as plant defensins, represents one typical family and comprises low molecular mass cysteine-rich proteins, usually cationic and distributed in different plant tissues. Here, we report the purification and characterization of a novel γ-thionin from cowpea seeds (Vigna unguiculata), named Cp-thionin II, with bactericidal activity against Gram-positive and Gram-negative bacteria. Once the primary structure was elucidated, molecular modelling experiments were used to investigate the multimerization and mechanism of action of plant γ-thionins. Furthermore, Cp-thionin II was also localized in different tissues in cowpea seedlings during germination in contrasting conditions, to better understand the plant protection processes. The use of plant defensins in the construction of transgenic plants and also in the production of novel drugs with activity against human pathogens is discussed.