To properly design and investigate new antibacterial drugs a detailed description of the organization of bacterial membrane is highly important. Therefore in this work we performed a comprehensive characteristic of the Langmuir monolayers composed of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) mixed in a wide range of composition and treated as an artificial cytoplasmic layer of bacterial membrane. To obtain detailed information on the properties of these films we combined the analysis of the surface pressure–area curves with the surface potential measurements, Brewster Angle Microscopy studies and Grazing Incidence X-ray Diffraction experiments. It was found that the investigated phospholipids mix nonideally in the monolayers and that the most favorable packing of molecules occurs at their equimolar proportion. This is directly connected with the formation of hydrogen bonds between both types of molecules in the system. All the collected experimental data evidenced that dipalmitoylphosphatidylethanolamine (DPPE) and dipal- mitoylphosphatidylglycerol (DPPG) form highly ordered associates of fixed (DPPE:DPPG 1:1) stoichiometry. The obtained results allow one to conclude a nonuniform distribution of lipids in bacterial membranes and the existence of domains composed of the investigated phospholipids. The latter seems to be of great impor- tance in the perspective of further studies on the mechanism of action of antibacterial agents.