Cystine-based gemini surfactants with dodecyl, tetradecyl, hexadecyl, and octadecyl hydrocarbon chains were synthesized, and their interactions with unsaturated (soy phosphatidylcholine, SPC)/saturated (hydrogenated SPC, HSPC) soy phosphatidylcholines in the forms of a monolayer and a model liposome were estimated for different combinations of the components in the mixed systems. Studies of Langmuir monolayers at the air aqueous buffer interface revealed condensation of the monomolecular films with the addition of surfactants. The effect of surfactants decreased according to the following order: octadecyl > hexadecyl > tetradecyl > dodecyl homologs. The nonideal mixing between the components was estimated using the deviation of the experimental molecular area from the ideal area per molecule. The excess molecular area increased with the increase in the surfactant chain length and phospholipid saturation. The 50 mol % mixture of cystine derivatives and phospholipids formed thermodynamically stable monolayers. The surfactants increased the rigidity of SPC monolayers and decreased that of HSPC monolayers, as observed by the studies of surface dialational rheology. The film structure at the air water interface could differentiate the SPC- and HSPC-comprising systems through the formation of organized regions, especially at a higher surface pressure. The constriction of surfactant/phospholipid hybrid vesicles was observed with an increase in the length of surfactant hydrocarbon chains. The negative zeta potential of vesicles took the highest values and did not change with time for 20 and 50 mol % surfactant. The spherical shape of the vesicles was confirmed by transmission electron microscopy. Differential scanning calorimetry revealed an increase in fluidity of HSPC bilayers and rigidity of SPS bilayers under the influence of surfactants. These effects were confirmed by fluorescence spectroscopy. All of the vesicle formulations were found to be nontoxic from the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide assay, suggesting their potential as a novel membranous system for the delivery of drugs, genetic materials, vaccines, and other therapeutic agents.