Antimicrobial peptides (AMPs) seem to be promising alternatives to common antibiotics, which are facing increasing bacterial resistance. Among them are the cysteine-stabilized αβ defensins. These peptides are small, with a length ranging from 34 to 54 amino acid residues, cysteine-rich and extremely stable, normally composed of an α-helix and three β-strands stabilized by three or four disulfide bonds and commonly found in several organisms. Moreover, animal and plant CSαβ defensins present different specificities, the first being mainly active against bacteria and the second against fungi. The role of the CSαβ-motif remains unknown, but a common antibacterial mechanism of action, based on the inhibition of the cell-wall formation, has already been observed in some fungal and invertebrate defensins. In this context, the present work aims to group the data about CSαβ defensins, highlighting their evolution, conservation, structural characteristics, antibacterial activity and biotechnological perspectives.