Modification of the major bovine whey protein, β-lactoglobulin (β-LG) by 3-hydroxyphthalic anhydride (3HP) leads to the generation of a potent inhibitor of infection by human immunodeficiency virus (HIV) types 1 and 2, designated 3HP-β-LG. 3HP-β-LG also has antiviral activity against herpesviruses, albeit at concentrations exceeding those required for inhibition of HIV-1 infection. The topical application of 3HP-β-LG to decrease the rate of sexual transmission of HIV and other sexually transmitted viruses worldwide is being considered. Results presented here: (i) define the conditions for chemical modification of β-LG by 3HP, resulting in 3HP-β-LG with optimum anti-HIV-1 activity; (ii) show that β-LG, prior to chemical modification, or 3HP-β-LG can be exposed to the elevated temperatures used to pasteurize milk without adversely affecting anti-HIV-1 activity; (iii) provide evidence that 3HP-β-LG is a more potent anti-HIV-1 compound than sulphated polysaccharides, other candidate compounds considered as prophylactic agents to prevent sexual transmission of HIV-1; and (iv) confirm that the primary target for 3HP-β-LG is CD4, although binding to the HIV-1 envelope protein gp120 was also observed and contributed to the antiviral activity of 3HP-β-LG.