Herpesviruses as herpes simplex virus 1 (HSV-1), remain a significant health challenge due to their ability to establish lifelong infections that current treatments cannot eradicate. These double-stranded DNA viruses replicate and store their genetic material within the nuclei of host cells. Similarly to all forms of life, humans have dsDNA nucleases (DNases) that cut dsDNA to repair, edit, or eliminate unwanted genetic material. However, these large viruses are known to harbour a pro-viral arsenal of proteins, some of which may inactivate host DNases to protect the viral DNA. This renders the antiviral potential of DNases elusive and complicates definitive antiviral strategies. In this study, we aimed to characterize the susceptibility of human DNases to HSV-1 infection through computational analysis and experiments in primary cells. Our findings indicate that several DNases are predicted to interact with HSV-1 proteins, which is supported by confocal microscopy evidencing alterations in subcellular localization, as well as changes in protein levels observed via immunoblotting. Notably, we identified band shifts likely indicative of post-translational modifications (PTMs) driven by infection. Additionally, native immunoprecipitation coupled with mass spectrometry revealed novel interactions between host and viral proteins, shedding light on the complex interplay between HSV-1 and the host's DNase response.