Cationic antimicrobial peptides (CAMPs) are essential elements of immunity in higher organisms. These peptides are capable of exerting a direct antimicrobial effect through a process that appears to at least in part involve interaction between the peptides and the bacterial membrane, which ultimately leads to membrane disruption. The lipids and other components that comprise cellular membranes contain chiral centers with defined stereochemistry that CAMPs may encounter as they interact with the membrane. The significance of membrane and peptide chirality is not well defined. Helical CAMPs provide an attractive tool for addressing these questions. They adopt a helical conformation when they interact with bacterial membranes, but in the absence of the influence of negatively charged bacterial membranes, they assume a relatively unstructured random coil. Formation of an amphipathic helix is usually essential to the antimicrobial mechanism employed by these peptides. Accordingly, we have used a pair of short helical CAMP enantiomers, with sequences based on that of the Naja atra cathelicidin, to study how peptide stereochemistry impacts peptide-lipid interactions. We have employed several biophysical techniques to characterize the interactions between chiral membranes and the D- and L- forms of antimicrobial peptides, including circular dichroism to reveal the effect of membranes on peptide secondary structure and electron paramagnetic resonance to assess the effect of peptides on spin-labeled lipids. The biophysical studies are complemented by functional assays of the antimicrobial activities of each peptide.