Crystal structures of amphiphilic molecules should serve as a convenient basis to explore the molecular associations and intermolecular interactions in the corresponding Langmuir layers and Langmuir-Blodgett (LB) films. However, the difficulty of growing single crystals of such molecules often precludes such investigations. We present, in this paper, the crystal structure investigation and LB film study of new amphiphiles based on the 4-nitroaniline moiety of interest in quadratic nonlinear optical applications. A molecular-level explanation of the general instability of monolayers of these amphiphiles is proposed based on near neighbor interactions observed in the crystals. An approach to stabilize them through the formation of composite bilayers with the phospholipid molecule, distearoylphosphatidylcholine (DSPC), is developed. The complex pressure-area isotherms of the composites and their transformation through multiple isocycles are interpreted using the molecular structure and conformation from single crystal analysis. Electronic absorption spectroscopy of the solution, solid state, and LB films complemented by semiempirical quantum chemical computations allows us to model the intermolecular interactions in the LB film. This study demonstrates a novel approach to correlating the 3-dimensional and quasi-2-dimensional assembly of molecules in crystals and Langmuir/LB films.