We report in this work the isotherms of cholesterol and stearic acid at the air-water interface modified by different chitosans (chitosan chloride, hydrophobic modified chitosan, and medium and high molecular weight chitosans) in the aqueous subphase. The Langmuir-Blodgett films of the complexes cholesterol - chitosan and stearic acid-chitosan are analyzed by atomic force microscopy (AFM), and a molecular simulation was performed to visualize the chitosan-lipid interactions. Strong modifications are obtained in the isotherms as a result of the chitosan interactions with cholesterol and stearic acid at the air-water interface. These modifications were dependent on the type and concentration of chitosan. Severe modifications of all phases were noticed with larger molecular areas, and the observed changes in the compressional modulus were dependent on the type of chitosan used. The complexes of chitosan-stearic acid were more flexible than the ones of chitosan-cholesterol. The AFM images demonstrated that chitosan was disaggregated by the cholesterol and stearic acid interactions producing more homogeneous surfaces in some cases. The hydrophobic chitosan showed more affinity with stearic acid, while both medium and high molecular weight chitosans produced homogeneous surfaces with cholesterol. The simulated chitosan chains interacting with cholesterol and stearic acid demonstrated the possibility of specific sites of electrostatic bonds between these molecules. Adsorption of cholesterol on the different powdered chitosans, performed by HPLC, showed that the medium and high molecular weight chitosans could retain higher proportions of cholesterol compared with the other analyzed samples.