Toll-like receptors (TLRs) are pattern recognition transmembrane proteins that play an important role in innate immunity. In particular, TLR7 plays a role in detecting nucleic acids derived from viruses and bacteria. The huge number of pathologies in which TLR7 is involved has led to an increasing interest in developing new compounds targeting this protein. Several conjugation strategies were proposed for TLR7 agonists to increase the potency while maintaining a low toxicity. In this work, we focus the attention on two promising classes of TLR7 compounds derived from the same pharmacophore conjugated with phospholipid and polyethylene glycol (PEG). A multidisciplinary investigation has been carried out by molecular dynamics (MD), dynamic light scattering (DLS), electron paramagnetic resonance (EPR), and cytotoxicity assessment. DLS and MD indicated how only the phospholipid conjugation provides the compound abilities to self-assemble in an orderly fashion with a maximal pharmacophore exposition to the solvent. Further EPR and cytotoxicity experiments highlighted that phospholipid compounds organize in stable aggregates and well interact with TLR7, whereas PEG conjugation was characterized by poorly stable aggregates at the cells surface. The methodological framework proposed in this study may be used to investigate, at a molecular level, the interactions generally occurring between aggregated ligands, to be used as drugs, and protein receptors.