Lactoferrin (LF) is a multifunctional glycoprotein, which plays an important role in immune regulation and defense mechanisms against bacteria, fungi, and viruses. Upon peptic digestion of LF, a peptide called lactoferricin (Lfcin) is generated. Lfcin corresponds to the N-terminal part of the protein. In this study we investigated the antiviral activity of bovine and human Lfcin against Herpes simplex virus (HSV)-1 and HSV-2. The 50% effective concentrations (EC(50)) for LF and Lfcin against several clinical isolates of HSV-1 and HSV-2, including acyclovir (ACV)-resistant strains, were determined. We further evaluated the effect of the combination of either LF or Lfcin with ACV against HSV-1 and HSV-2. Synergy was observed between both LF or Lfcin in combination with ACV against the HSV laboratory strains. The 50% effective concentration (EC(50)) for ACV and LF or Lfcin, when combined with ACV, could be reduced by two- to sevenfold compared to the EC(50) when the drugs were used alone.