Plant defensins are cationic peptides that are ubiquitous within the plant kingdom and belong to a large superfamily of antimicrobial peptides found in several organisms collectively called defensins. The primary structure of these peptides includes 45 to 54 amino acid residues with considerable sequence variation. At the level of three-dimensional structure, they are small and globular, composed of three anti-parallel β-sheets and one α-helix, which is highly conserved among these peptides. The three-dimensional structure is stabilized by four disulfide bridges formed by eight strictly conserved Cys residues. Two of these bridges compose the Cys-stabilized α-helix β-strand motif, which is found in other peptides with biological activities. Plant defensins present numerous biological activities, such as inhibiting protein synthesis, ion channel function and α-amylase and trypsin activity; impairing microbial, root hair and parasitic plant growth; mediating abiotic stress and Zn tolerance; altering ascorbic acid redox state; stimulating sweet taste sensation; serving as epigenetic factors; affecting self-incompatibility; and promoting male reproductive development. Some of these biological activities, such as microbial growth inhibition and sweet taste induction, coupled with a scaffold that provides these peptides with incredible physicochemical resistance to harsh environments and the potential for simple amino acid substitution, raise the opportunity to improve the function of defensins or introduce new activities, endowing these peptides with great biotechnological and medical significance. This review will cover the biological activities and roles of plant defensins and will focus on their application in the field of biotechnology.