In this work, we studied the effects of three cryoprotectors – ethylene glycol, dimethyl sulfoxide and sucrose – on the compression isotherms of egg yolk Langmuir monolayers both in the presence and in the absence of cholesterol in the monolayer. The influence of calcium ions from the subphase affecting the effectiveness of cryoprotection on π-A isotherms is also examined. In addition, the elastic properties of the obtained monolayers are investigated by calculation and comparison the compression modulus of the monolayer. The scientific novelty of the work is in consideration of a complex biosimilar system (an egg yolk monolayer, cholesterol and their mixtures) on the surface of the aqueous solution of the nutrient mixture and obtaining information about the specific interaction of different cryoprotectors with lipid membranes. We found that when calcium ions and cryoprotectors are simultaneously added to the subphase, they block each other's influence on the lipid monolayer and reduce the effectiveness of cryoprotection. Cholesterol in the yolk in a ratio of 1:50 m m−1 changes the properties of the monolayer, which leads to increased action of cryoprotectors. Also, for the first time, the effect of a significant increase in surface pressure (by ∼20 mN m−1) was detected when cryoprotectors were added to the system under consideration. This effect can serve as an indicator of the effectiveness of membrane dehydration by cryoprotectors and can be used to find the most effective and safe cryoprotector compositions. The obtained data can provide important recommendations for the development of cryoprotective media for cell freezing. Since the study of the mechanisms of calcium interaction (the most important signaling cation) with biological membrane and membrane-like systems is important for understanding the various effects caused by medicinal and biologically active drugs at the cellular level, the study is of interest for various fields of biophysics and biomedicine.