Adsorption, self-organization, and mechanical properties of different fatty acid layers under different confinement states have been investigated as a function of the presence and conformation of one unsaturation in their aliphatic chain. In situ characterization, at the molecular level, was performed with the ATLAS molecular tribometer, in terms of rheology, forces, and thickness of confined fluid. The results demonstrate that the fatty acids adsorb on the surfaces by weak interactions and form viscoelastic films with a thickness of about 15 Å on each surface. The adsorption kinetics, the packing of the self-assembled monolayers, and the coverage rate depend on the molecular architecture of the fatty acids and lead to various mechanical behaviors under confinement.