The search for new antibiotic agents is continuous, reflecting the continuous emergence of antibiotic-resistant pathogens. Among the new agents are the antimicrobial peptides (AMPs), which have the potential to become a leading alternative to conventional antibiotics. Studies for the mechanisms of action of the naturally occurring parent peptides can provide the structural and functional information needed for the development of effective new antibiotic agents. We therefore characterized pseudin-2, an AMP isolated from the skin of the South American paradoxical frog Pseudis paradoxa. We found that pseudin-2 organized to an aggregated state in aqueous solution, but that it dissociated into monomers upon binding to lipopolysaccharide (LPS), even though it did not neutralize LPS in Gram-negative bacteria. In addition, pseudin-2 assumed an α-helical structure in the presence of biological membranes and formed pores in both bacterial and fungal membranes, through which it entered the cytoplasm and tightly bound to RNA. Thus, the potent antimicrobial activity of pseudin-2 likely results from both the formation of pores capable of collapsing the membrane potential and releasing intracellular materials and its inhibition of macromolecule synthesis through its binding to RNA.