Liposomes are self-assembled lipid bilayer nanostructures with an inner aqueous core used for diagnostic signal amplification, drug delivery, and as biomimics. Small molecules and proteins are typically encapsulated. While the lipid composition is used to control liposome and surface characteristics, overlooked is the effect entrapped molecules may have on the outer surface through interface activity. Here, it is demonstrated how different dyes not only distribute between the aqueous core and bilayer but also significantly affect the outer surface chemistry thus influencing interactions with reaction partners and sample matrices. Specifically, IR-783, sulforhodamine B (SRB), and 1,3,6,8-pyrenetetrasulfonic acid (PTSA) are encapsulated in liposomes of standard bioanalytical composition. Spectroscopic and small-angle X-ray scattering data indicate interactions of IR-783 with the liposome membrane and its strong influence on the bilayer structure. Increasing SRB concentrations show potential adsorption at liposome bilayer surfaces, whereas PTSA does not interact with the bilayer itself. Surprising is the correlating effect on biological systems discovered through the complement system as a model. Liposomes incubated with serum reveal complement protein interaction with the liposome surface depending on the dye and not only on the lipid composition. This study emphasizes the need for careful selection of both lipid and encapsulant formulations in any biological application.