Naturally occurring substances with antimicrobial activity can serve as a starting point for the rational design of new drugs to treat infectious diseases. Here, we screened a library of peptides derived from human hemofiltrate for inhibitory effects on human cytomegalovirus (CMV) infection. We isolated a previously unknown derivative of the neutrophil-activating peptide 2, which we termed CYVIP, for CMV-inhibiting peptide. The peptide blocked infection with human and mouse CMV as well as with herpes simplex virus type 1 in different cell types. We found that CYVIP interferes with virus attachment to the cell surface, and structure-activity relationship studies revealed that positively charged lysine and arginine residues of CYVIP are essential for its inhibitory activity. The N-terminal 29 amino acids of the peptide were sufficient for inhibition, and substitution with an acidic residue further improved its activity. The target structure of CYVIP on the cell surface seems to be the sulfate residues of heparan sulfate proteoglycans, which are known to serve as herpesvirus attachment receptors. Our data suggest that O-sulfation of heparan sulfate is required for binding of CYVIP, and furthermore, that the initial interaction of CMV particles with cells takes place preferentially via 6-O-linked sulfate groups. These findings about CYVIP's mode of action lay the basis for further development of antivirals interfering with attachment of CMV to cells, a crucial step of the infection cycle.