The extensive search for alternative therapeutics against microbial pathogens has led to the discovery of cationic peptides as new anti-infectives with a novel mode of action. Particular interest has been devoted to small linear peptides that can be efficiently made by chemical synthesis at competitive costs. The most promising originate from a large family of short, naturally occurring peptides found in the skin of amphibia of Rana genus, i.e. the temporins. This review is mainly focused on the recent structure-function studies of the earliest known temporin isoforms (TA, TB and TL) and their potential clinical role as novel antimicrobial agents. The development of novel antibiotics is an urgent public health concern due to the increased resistance of microorganisms to conventional antibiotics, particularly in the hospital setting.