Cobra venom cardiotoxins (CVCs) can translocate to mitochondria to promote apoptosis by eliciting mitochondrial dysfunction. However, the molecular mechanism(s) by which CVCs are selectively targeted to the mitochondrion to disrupt mitochondrial function remains to be elucidated. By studying cardiotoxin from Naja mossambica mossambica cobra (cardiotoxin VII4), a basic three-fingered S-type cardiotoxin, we hypothesized that cardiotoxin VII4 binds to cardiolipin (CL) in mitochondria to alter mitochondrial structure/function and promote neurotoxicity. By performing confocal analysis, we observed that red-fluorescently tagged cardiotoxin rapidly translocates to mitochondria in mouse primary cortical neurons and in human SH-SY5Y neuroblastoma cells to promote aberrant mitochondrial fragmentation, a decline in oxidative phosphorylation, and decreased energy production. In addition, by employing electron paramagnetic resonance (EPR) and protein nuclear magnetic resonance (1H-NMR) spectroscopy and phosphorescence quenching of erythrosine in model membranes, our compiled biophysical data show that cardiotoxin VII4 binds to anionic CL, but not to zwitterionic phosphatidylcholine (PC), to increase the permeability and formation of non-bilayer structures in CL-enriched membranes that biochemically mimic the outer and inner mitochondrial membranes. Finally, molecular dynamics simulations and in silico docking studies identified CL binding sites in cardiotoxin VII4 and revealed a molecular mechanism by which cardiotoxin VII4 interacts with CL and PC to bind and penetrate mitochondrial membranes.