Molecular assemblies containing phospholipids and conjugated polydiacetylene lipids exhibit unique biochromatic properties and have attracted increasing interest in recent years as potential bio- and chemosensors. We present a detailed study of the properties of mixed films formed at the air-water interface, which consist of phospholipid molecules and diacetylene lipids. The organization of the films has been characterized by surface pressure-area isotherms. Application of atomic force microscopy, polarized optical microscopy, and UV-vis spectroscopy provides further insight into the structures and interactions of the film components. The data indicate that the two constituents in the film are miscible at low surface pressure, while segregation of phospholipid and polymer domains occurs at higher surface pressures. The distribution and interactions between the diacetylene and phospholipid domains additionally depends on the molar fraction of phospholipid in the film. Characterization of the structural properties of the polydiacetylene domains in the films points to a formation of organized trilayer and multilayer phases at high surface pressures and high diacetylene concentrations.