The molecular organization and the domain morphology of an anionic matrix dimyristoyl−phosphatidic acid (DMPA) as influenced by the presence of a cationic water-soluble methylene blue (MB) have been studied by using Brewster angle microscopy and reflection spectroscopy at the air−water interface. Mixed monolayers of DMPA:MB, molar ratios 5:1 and 1:1, were formed by the cospreading method. BAM images show particular domain morphologies as a function of the MB density in the mixed film: circular domains for DMPA:MB = 5:1, and domains with hexagonal shape for DMPA:MB = 1:1. The reflection measurements during the compression process of the mixed films reveal a blue shift of the visible band with respect to that of the MB monomer. This blue shift is larger for the DMPA:MB = 5:1 in comparison with that for the 1:1 monolayer, despite lower MB density. By using the extended dipole model, we demonstrate that such blue shift is due to the formation of a 2D hexagonal network of MB molecules underneath the DMPA monolayer in the mixed 1:1 film, while it is due to infinite linear aggregates in the 5:1 monolayer. BAM domain textures have been simulated by using the Fresnel equations for biaxial anisotropic materials. The results fit correctly the predictions of the extended dipole model.