Achieving a high nutritional value of food often involves fortifying microorganisms (such as bacteria and yeast) used in baking and dairy industry with essential elements. The aim of this study was to investigate the effect of a pulsed electric field (PEF) on the penetration and accumulation of Ca2+ and Mg2+ ions into model membranes of the food-grade yeast Saccharomyces cerevisiae. Simplified model membranes (monolayers and liposomes) were constructed using the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). The Langmuir monolayer technique, dynamic light scattering (DLS) and microelectrophoresis were employed to characterize the physicochemical properties of the model membranes investigated. The results showed significant molecular-level differences in the interactions of the selected cations with lipid monolayers and bilayers in liposome structures. Both cations deeply penetrated the membrane’s hydrophilic region, yet two competing effects were evident: expansion induced by hydrated Mg2+ and condensation driven by Ca2+ bridging. Furthermore, the application of PEF increased the concentration of ions absorbed by the liposomes. Specifically, optimized PEF parameters resulted in cation accumulation within the model membranes, ranging from 6 to 13%. This finding correlates well with the increased Ca2+ and Mg2+ uptake observed in real yeast cells, providing a deeper understanding of the cell membrane-environment interface and the underlying processes.