Native and chemically derivatized proteins purified from serum and milk were assayed in vitro to assess their inhibiting capacity on the cytopathic effect of human immunodeficiency virus (HIV)-1 and human cytomegalovirus (HCMV) on MT4 cells and fibroblasts, respectively. Only native and conformationally intact lactoferrin from bovine or human milk, colostrum, or serum could completely block HCMV infection (IC₅₀ = 35–100 µg/mL). Moreover, native lactoferrin also inhibited the HIV-1-induced cytopathic effect (IC₅₀ = 40 µg/mL). When negatively charged groups were added to lactoferrin by succinylation, there was a 4-fold stronger antiviral effect on HIV-1, but the antiviral potency for HCMV infection was mostly decreased. Lactoferrin likely exerts its effect at the level of virus adsorption or penetration (or both), because after HCMV penetrated fibroblasts, the ongoing infection could not be further inhibited.