Thermodynamic and structural aspects of the monolayer film formation of 3-monoacyl-sn-glycerol homologous series with the alkyl chain length n = 7–18 at the air/water interface are considered. The calculations are carried out within the framework of the semiempirical quantum chemical method PM3 and supported experimentally using surface pressure-area per molecule (π–A) isotherms, Brewster angle microscopy (BAM), as well as grazing incidence X-ray diffraction analysis (GIXD). A conformational analysis of 3-monoacyl-sn-glycerol monomers reveals three stable conformations of monomers with Monomer 1 as the possible basis for the construction of the most energetically advantageous structures of small clusters, which subsequently can form a 2D monolayer. Optimized tetramer structure of 3-monoacyl-sn-glycerol is considered as a unit cell of the 2D film with geometric parameters: a= 4.82 Å, b= 4.92 Å, and the angle between them θ = 91°. The molecular tilt angle of surfactant molecules with respect to the normal to the interface is 23.5°. These structural parameters are in good agreement with existing experimental data. The spontaneous clusterization threshold of 3-monoacyl-sn-glycerol at the air/water interface is 16 carbon atoms in the alkyl chain at standard temperature. The temperature dependences of the Ac area per film molecule at the beginning of the liquid-expanded–liquid-condensed (LE–LC) phase transition are analyzed. The values of the Ac change per 1 °C for 3-monoacyl-sn-glycerol are commensurable with corresponding values of other surfactant classes confirming the dominating role of intermolecular CH···HC interactions in the clusterization energy of surfactants.