Nanoplasmonic sensing (NPS), based on localized surface plasmon resonance, with sensors composed of glass covered with golden nanodisks and overlaid with a SiO2 coating was applied in this study. Egg phosphatidylcholine (eggPC), being an easily accessible membrane-forming lipid, was used for preparation of biomimicking membranes. Small unilamellar vesicles with an approximate hydrodynamic diameter of 30 nm, formed by sonication in 4-(2-hydroxyethyl)piperazine-l-ethanesulfonic acid buffer, were adsorbed within 10 min on the sensor surface either as intact vesicles or as a planar bilayer. The adsorbed biomembrane systems were further utilized for interaction studies with four different well-known surfactants (negatively and positively charged, zwitterionic, and nonionic) and each surfactant was tested at concentrations below and above the critical micelle concentration (CMC). Our results allowed the evaluation of different NPS patterns for every particular supported membrane system, surfactant, and its concentration. The most significant effect on the membrane was achieved upon the introduction of zwitterionic surfactant micelles, which in fact completely solubilized and removed the lipid membranes from the sensor surface. Other surfactant micelles interacted with the membranes and formed mixed structures remaining on the sensor surface. The studies performed at the concentrations below the CMCs of the surfactants showed that different mixed systems were formed. Depending on the supported membrane system and the type of surfactant, the mixed systems indicated different formation kinetics. Additionally, the final water rinse revealed the stability of the formed systems. To investigate the effect of the studied surfactants on the overall surface charge of the biomembrane, capillary electrophoresis (CE) experiments were carried out in parallel with the NPS analysis. The electroosmotic flow mobility of an eggPC-coated fused silica capillary was used to measure the total surface charge of the biomembrane after its treatment with the surfactants. Our results indicated in general good correlation between CE and NPS data. However, some discrepancies were seen while applying either zwitterionic or positively charged surfactants. This confirmed that CE analysis was able to provide additional data about the investigated systems. Taken together, the combination of NPS and CE proved to be an efficient way to describe the nature of interactions between biomimicking membranes and amphiphilic molecules.