Lipid membranes are crucial for cellular metabolism, and their correct function has been linked to a tight regulation of their structural and mechanical properties, such as viscosity. Fluorescent probes sensitive to the membrane’s environ- are being extensively used to investigate the membrane’s properties, yet there is currently a lack of understanding on how the lipid organization impacts the readout from these dyes. Here, we investigate this relationship by simultaneously characterizing the membrane’s viscosity and structural properties using a combination of X-Ray diffraction, together with environmentally-sensitive optical membrane probes and fluorescence lifetime imaging microscopy. Our results reveal a phase-dependent connection between the different membrane’s structural and mechanical parameters and give insight into the relationship between two widely used membrane probes with the structural descriptors of the lipid bi-layer. Such relationship is believed to dictate the lateral organization of lipid bilayers, including the presence of distinct lipid domains which have been traditionally ascribed to a difference in the membrane thickness of the lipid phases; yet we later demonstrate how such connection is not universal and can disrupted by the presence of line-active molecules. Our results show the capability of membrane dyes to directly report on the membrane’s molecular structure – after appropriate calibration – and highlight the need of multiple orthogonal characterization strategies for a proper understanding of the membrane’s properties.