Human cytomegalovirus (HCMV) and Herpes Simplex Virus (HSV-1) are double-stranded DNA viruses of the herpesvirus family. Herpesviruses are ubiquitous viruses that establish life-long latency with the potential to reactivate resulting in a range of diseases. Galectins are a family of cellular proteins characterised by the presence of a carbohydrate recognition domain that facilitates their lectin functionality. Members of this widely expressed protein family have been implicated in modulating both anti-viral immunity and regulation of direct host-virus interactions. Previous studies from the Slobedman laboratory have established Galectin-9 was potently upregulated by the anti-viral cytokine IFN-β. However, the potential for galectins to directly modulate infection has not previously been studied in the context of HCMV or HSV-1. Infection studies utilising recombinant protein treatment, revealed Galectin-9, but not Galectin-1, as an anti-viral lectin that inhibited both HCMV and HSV-1 infection of primary human fibroblasts, and a human epithelial cell line, in a dose dependent manner. Further analysis established that Galectin-9 mediated inhibition of infection could be blocked by anti-Galectin-9 specific antibodies, and was carbohydrate recognition domain-dependent. Temperature-shift studies that separated the binding and entry stages of infection identified Galectin-9 specific inhibition as mediated primarily at the level of viral entry, and appeared to be dependent on binding to the virion rather than interacting with cellular ligands. This study provides the first evidence of a novel role for Galectin-9 as an anti-viral lectin that exerts a profound inhibitory effect on both HCMV and HSV-1 infection.