This review focuses on the first plant defense protein class described in literature, with growth inhibition activity toward pathogens. These peptides were named gamma-thionins or defensins, which are small proteins that can be classified into four main subtypes according to their specific functions. Gamma-thionins are small cationic peptides with different and special abilities. They are able to inhibit digestive enzymes or act against bacteria and/or fungi. Current research in this area focuses particularly these two last targets, being the natural crop plant defenses improved through the use of transgenic technology. Here, we will compare primary and tertiary structures of gamma-thionins and also will analyze their similarities to scorpion toxins and insect defensins. This last comparison offers some hypothesis for gamma-thionins mechanisms of action against certain pathogens. This specific area has benefited from the recent determination of many gamma-thionin structures. Furthermore, we also summarize molecular interactions between plant gamma-thionins and fungi receptors, which include membrane proteins and lipids, shedding some light over pathogen resistance. Researches on gamma-thionins targets could help on plant genetic improvement for production of increased resistance toward pathogens. Thus, positive results recently obtained for transgenic plants and future prospects in the area are also approached. Finally, gamma-thionins activity has also been studied for future drug development, capable of inhibit tumor cell growth in human beings.