Lactoferrin (LF) is a multifunctional glycoprotein, which plays an important role in immune regulation and defense mechanisms against bacteria, fungi, and viruses. Lactoferricin (Lfcin) is a potent antimicrobial peptide generated from the N-terminal part of LF by pepsin cleavage. In this study, we investigated the mechanisms of the anti-herpes simplex virus (anti-HSV) activity of LF and Lfcin. The results demonstrated that LF and Lfcin inhibited the entry of HSV into Vero cells. LF had no effect against HSV after the virus had entered the cells, while Lfcin exerted antiviral activity also after the initial binding of the virus to the host cell. The distribution of LF and Lfcin in the cells was investigated by immunogold-labeling and transmission electron microscope (TEM). LF was found mainly at the cell surface in cells expressing heparan sulphate. Lfcin was randomly distributed intracellularly. LF must be present at the cell surface to exert antiviral activity, while Lfcin exert its antiviral activity also when found mainly intracellularly. Both LF and Lfcin were dependent on the presence of heparan sulphate at the cell surface to exert their antiviral activity.