The peptide TAT-I24, composed of the 9-mer peptide I24 and the TAT (48-60) peptide, exerts broad-spectrum antiviral activity against several DNA viruses. The current model of the mode of action suggests a reduction of viral entry and also a possible interaction with the viral DNA upon virus entry. To further support this model, the present study investigates the DNA binding properties of TAT-I24. DNA binding was analysed by gel retardation of a peptide-complexed DNA, fluorescence reduction of DNA labelled with intercalating dyes and determination of binding kinetics by surface plasmon resonance. Molecular dynamics simulations of DNA-peptide complexes predict high-affinity binding and destabilization of the DNA by TAT-I24. The effect on viral DNA levels of infected cells were studied by real-time PCR and staining of viral DNA by bromodeoxyuridine. TAT-I24 binds double-stranded DNA with high affinity, leading to inhibition of polymerase binding and thereby blocking of de novo nucleic acid synthesis. Analysis of early steps of virus entry using a bromodeoxyuridine-labelled virus as well as quantification of viral genomes in the cells indicate direct binding of the peptide to the viral DNA. Saturation of the peptide with exogenous DNA can fully neutralize the inhibitory effect. The antiviral activity of TAT-I24 is linked to its ability to bind DNA with high affinity. This mechanism could be the basis for the development of novel antiviral agents.