Mixed monolayers of 1-monostearoyl-rac-glycerol (StGl) and 1-(12-hydroxy)monostearoyl-rac-glycerol (12OH-StGl) were studied by means of pressure−area (π−A) isotherms, Brewster angle microscopy (BAM) and grazing incidence X-ray diffraction (GIXD). Thermodynamic analysis of the π−A isotherms of the mixed films showed strong deviations from the ideal behavior at all compositions. Negative deviations from ideality are observed in the monolayers with low 12OH-StGl content. The 12OH-StGl molecules are homogeneously distributed in the StGl matrix. The inclusion of 12OH-StGl molecules decreases the pressure at which the NN → NNN tilt transition occurs. A tilted phase with an oblique lattice and chain tilt in an intermediate (I) direction is observed in the mixed monolayers with small xOH values. With increasing mole fraction of the 12OH-StGl component positive deviations from ideal behavior are observed. The averaged interactions between the like molecules of the pure substances dominate, and the mixed monolayer tends to phase separate. Continuous change in the shape of the condensed phase domains during their growth also suggests partial phase separation in the mixed monolayers with high content of 12OH-StGl. Two condensed phases, StGl-rich and 12OH-StGl-rich, with different lattices coexist in the mixed StGl/12OH-StGl monolayers. Partial phase separation in these films might be due to the different lattice symmetry of the condensed phases of StGl and 12OH-StGl monolayers. The formation of the hydrogen bonds between the aliphatic chains of the molecules is supposed to stabilize the 12OH-StGl-rich phase. The composition−pressure phase diagram for the mixed StGl/12OH-StGl monolayers is constructed according to the data obtained by the π−A isotherms, BAM and GIXD.