<p>The development of snake venom-based therapeutics and antivenoms is closely linked to understanding how snake venoms modulate the physicochemical properties of biological membranes. In this study, we investigated the effects of <i>Macrovipera lebetina</i> venom on the surface electrical characteristics of rat liver mitochondrial membranes in vitro. A combination of electrokinetic and mechanical techniques was employed, including measurements of mitochondrial electrophoretic mobility and the bending elasticity of phosphatidylcholine membrane models. Exposure to <i>Macrovipera lebetina</i> venom significantly increased the net surface charge of mitochondria, while adenosine triphosphatase (ATPase) activity remained unchanged. Morphological alterations and vesicle aggregation, observed via light scattering and fluorescence microscopy, indicated reduced binding of α-D-mannosyl and α-D-glucosyl residues to the outer mitochondrial membrane in the presence of venom. In addition, malondialdehyde assays revealed elevated levels of oxidized lipid species in venom-treated mitochondria. A pronounced softening of model phosphatidylcholine membranes was also observed, likely associated with lipid oxidation induced by the venom. It states that the effect of the venom of the Macrovipera lebetina on the surface electrical charge of rat liver mitochondria is being studied in vitro. The surface charge is a physicochemical characteristic of the mitochondrial membrane.</p> Graphical abstract <p></p>