Natural polycationic membrane-active peptides typically lack disulfide bonds and exhibit fusion, cell-penetrating, antimicrobial activities. They are mostly unordered in solution, but adopt a helical structure, when bound to phospholipid membranes. Structurally different are cardiotoxins (or cytotoxins, CTs) from cobra venom. They are fully β- structured molecules, characterized by the three-finger fold (TFF). Affinity of CTs to lipid bilayer was shown to depend on amino acid sequence in the tips of the three loops. In the present review, CT-membrane interactions are analyzed through the prism of data on binding of the toxins to phospholipid liposomes and detergent micelles, as well as their structural and computational studies in membrane mimicking environments. We assess different hydrophobicity scales to compare membrane partitioning of various CTs and their membrane effects. A comparison of hydrophobic/hydrophilic properties of CTs and linear polycationic peptides provides a key to their biological activity and creates a fundamental basis for rational design of new membrane-interacting compounds, including new promising drugs. For instance, from the viewpoint of the data obtained on model lipid membranes, cytotoxic activity of CTs against cancer cells is discussed.