Pore-forming cytolysins damage the integrity of phospholipid membranes and cause lysis and cell death. Activation of cholesterol-dependent cytolysins (CDCs) occur in the presence of cholesterol in plasma membranes, however, some CDCs require additional ligands to exert cytotoxicity. Vaginolysin (VLY) from Gardnerella vaginalis and intermedilysin (ILY) secreted by Streptococcus intermedius belong to the group of CDCs whose activity is modulated by the human complement regulatory protein CD59. While the activity of VLY, though significantly lower, was observed in the absence of CD59, ILY is believed to be strictly CD59 dependent. Using a highly sensitive membrane damage detection technique based on tethered bilayer membrane (tBLM) technology, we assessed the dielectric damage of phospholipid membranes by three CDCs: VLY, ILY, and pneumolysin (PLY) - secreted by Streptococcus pneumoniae, known to not be dependent on CD59 - and found that, in the absence of CD59, membrane damage was triggered by all three. At constant protein concentration and membrane composition, protein activities decreased in the sequence PLY>VLY>ILY. The activities were sensitive to the concentration of cholesterol in the membranes, as well as the phospholipid composition. In particular, charged components such as phosphatidylserines inhibited PLY and ILY, while the activity of VLY remained unaffected. Components that facilitate lipid raft formation, such as sphingomyelin, inhibited the dielectric damage of tBLMs by all three CDCs, with significantly higher inhibition for ILY. Our data indicate that all studied CDCs are capable of damaging the dielectric barrier of cholesterol-containing model phospholipid membranes. In real conditions, however, surface cholesterol may not be readily accessible for protein binding, so additional ligands such as CD59 are utilized, to recruit sufficient amounts of monomer proteins for pore formation.