Lactoferrin (Lf) is a multifunctional glycoprotein that plays an important role in immune regulation and defence mechanisms against bacteria, fungi and viruses. Bovine lactoferrin (bLf) has been recognized as a potent inhibitor of human herpetic viruses, such as cytomegalovirus and herpes simplex virus type 1 and 2. BLf has been found to prevent viral infection by binding to heparan sulphate containing proteoglycans that also act as cell receptors for herpetic viruses. In this study we further investigated the inhibiting activity of bLf against herpes simplex virus type 1 (HSV-1) in Green Monkey Kidney (GMK) cells and found that, in addition to the viral adsorption step, bLf also targets the HSV-1 entry process and cell-to-cell viral spread. Our study showed that the inhibition of HSV-1 infectivity by bLf is dependent on its interaction with specific structural viral proteins. Apart from the prevention of early phases of viral infection, cell-to-cell spread inhibition activity of HSV-1 by bLf confirmed that this protein is an outstanding candidate for the treatment of herpetic infections since it would offer the advantage to prevent also viral infections caused by cell-associated virus.