The snake cytotoxin (SCT) family is a highly basic three-finger toxin stabilized by anti-parallel β-pleated sheets. Its three-finger functional loops interact with the cell membrane and induce cell death. However, contradictory findings have been reported regarding SCT-induced cell death. Thus, this study aimed to investigate the membrane perturbing effects of SCT using experimental and computational approaches. Extracellular lactate dehydrogenase (LDH) activity and calcein-AM fluorescent signals were quantified to determine the degree of membrane permeabilization following exposure to SCT. Then, a coarse-grained molecular dynamics (CGMD) simulation of SCT was modeled on a membrane system composed of an equal percentage of palmitoyloloeyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylserine (POPS), and sphingomyelin (DPSM). Extracellular LDH activity calcein-AM fluorescence staining revealed that SCT induced concentration-dependent membrane permeabilization without total cytolysis. On the other hand, the findings of CGMD simulations indicated that only one functional loop of SCT interacted with the membrane lipid bilayer system without complete penetration into the lipid bilayer. POPS was presumably the main phospholipid involved in direct interaction with SCT. Furthermore, the minimum distance of the lipid bilayer from SCT remained unchanged after 2 µs of simulation, suggesting probable pore formation without total membrane lysis. Altogether, the findings concluded that SCT induced loss of membrane integrity which occurred independently of total cytolytic effects.