In order to provide tools for investigations of amphotericin B ion channels, new conjugates bearing a calix[4]arene scaffold covalently linked to four amphotericin B molecules were synthesized. These macromolecules adopt a cone conformation that mimics the structure of a transmembrane pore. The antifungal activity of the conjugates 3 and 4 was superior or similar to that of native amphotericin B, with minimal inhibitory concentration values of 0.10 and 0.25 μM, respectively. Furthermore, the hemotoxicity of the new conjugates was considerably lower (at least 10 times) than the hemotoxicity of monomeric amphotericin B. Finally, the formation of ion channels in the lipid bilayer by the amphotericin B tetramer was monitored by measuring the K⁺ efflux from various liposomes.