In this study, the effect of polyols, erythritol, xylitol, mannitol, on a model membrane systems composed of DMPC was investigated using differential scanning calorimetry and Fourier transform infrared spectroscopy. Generally, it is considered that polyols possess strong hydrophilic properties, and either does not interact with the hydrophobic environment at all, or these interactions are very weak. To better understand the mutual interactions between polyols and the lipid system, the Langmuir technique was used to examine the molecular organization of monolayers and to calculate their thickness in the presence of polyols at the subphase. The detailed description of the interactions between polyols and DMPC molecules was complemented by the analysis of the morphology of monolayers with the application of Brewster angle microscopy. From ATR FTIR, the significant spectral shift is observed only for the PO2− stretching band, which correlates strongly with the polyol chain-length. The longer the polyol chain, the weaker the observed interactions with lipid molecules. The most important findings, obtained from thickness measurements, reveal that short-chain polyols may prevent the formation of bilayers by the DMPC molecules under high surface pressure. The changes in the organization of DMPC monolayers on the surface, as visualized by Brewster angle microscopy, showed that the domains observed for phospholipid film spread on pure water differ substantially from those containing polyols in the subphase.