Approximately 22% of moderately to severely affected atopic dermatitis (AD) patients have a history of eczema herpeticum, a disseminated rash primarily caused by herpes simplex virus type 1 (HSV-1). Reduced activity of antimicrobial peptides may contribute to the increased susceptibility of AD patients to HSV-1. We previously demonstrated that the antimicrobial protein RNase 7 promotes self-DNA sensing by human keratinocytes, thereby strengthening their resistance to HSV-1 infection. Here, we investigated the effects of RNase 7 in the absence of exogenously added costimulatory DNA. Recombinant RNase 7 restricted HSV-1 gene expression, genome replication, and plaque formation in human keratinocytes. It decreased HSV-1 immediate-early transcripts independently of the induction of interferon-stimulated genes. Its main effect was on intracellular infection processes and not on extracellular virions or virus binding to cells. RNase 7 reduced the amount of cell-associated capsids and the HSV-1 envelope glycoprotein D at 3 but not at 0.5 h post-infection, suggesting that it promoted the degradation of incoming viral particles. Our data show that RNase 7 directly restricts HSV-1 infection of human keratinocytes, suggesting that it limits HSV-1 spread in the skin, and thereby disseminated infection. Previously described mechanisms that reduce RNase 7 activity in the lesional skin of AD patients may increase their susceptibility to eczema herpeticum.