An amphipathic α-helical structure is considered to be a prerequisite for the lytic activity of most short linear cytolytic polypeptides that act on both mammalian cells and bacteria. This structure allows them also to exert diverse pathological and pharmacological effects, presumably by mimicking protein components that are involved in membrane-related events. In this study D-amino acid-incorporated analogues (diastereomers) of the cytolysin pardaxin, which is active against mammalian cells and bacteria, were synthesized and structurally and functionally characterized. We demonstrate that the diastereomers do not retain the α-helical structure, which in turn abolishes their cytotoxic effects on mammalian cells. However, they retain a high antibacterial activity, which is expressed in a complete lysis of the bacteria, as revealed by negative staining electron microscopy. The disruption of the α-helical structure should prevent the diastereomer analogues from permeating the bacterial wall by forming transmembrane pores but rather by dissolving the membrane as a detergent. These findings open the way for a new strategy in developing a novel class of highly potent antibacterial polypeptides for the treatment of infectious diseases, due to the increasing resistance of bacteria to the available antibacterial drugs.