Acyclovir has been conjugated to the acyclic isoprenoid chain of squalene to form the squalenoyl–acyclovir prodrug. Its interaction with biomembrane models constituted by dimyristoylphosphatidylcholine (DMPC) monolayers has been studied by employing the Langmuir–Blodgett technique. The aim of the work was to gain information on the interaction of these compounds with phospholipid membranes. DMPC/acyclovir or squalenoyl–acyclovir prodrug mixed monolayers have been prepared at increasing molar fractions of the compound and the isotherm mean molecular area/surface pressure has been registered at 10 and 37 °C. Results reveal that the squalenoyl moiety enhances the affinity of acyclovir for the biomembrane model.