Photodynamic therapy (PDT) is widely used to treat different tumors and is rapidly developing approach to inhibit replication of fungi, bacteria and viruses. Herpes simplex virus 1 (HSV-1) is frequently used model to study effects of PDT on enveloped viruses. Although a number of photosensitizing compounds (PS) has been tested for the antiviral properties, the analyses are usually limited to assessing the reduction in virus yield, and thus the molecular mechanisms of photodynamic inactivation remain poorly understood. In our study, we investigated the antiviral properties of TMPyP3-C17H35, an amphiphilic porphyrin-based PS, and the molecular mechanisms of HSV-1 inhibition. We show that the expression of genes (immediate early, early and late genes) was strongly reduced in cells pretreated with sub-toxic concentrations of TMPyP3-C17H35, leading to a significantly reduced virus replication. These results indicate that treatment of cells with the compound triggers unexplored intrinsic antiviral defense mechanisms. Curiously, in experiments where cells were treated after the infection we observed a strong effect of TMPyP3-C17H35 on the virus yield only when cells were treated shortly (30min) after the infection. There are several plausible explanations for such effect, however, inefficiency of the compound to inhibit the virus once it has initiated its transcription, indicates incoming virions as the major target, probably by damaging internalized virus capsids, disrupting internalization to nucleus and at the same time triggering antiviral intrinsic immunity. Indeed, we show that the compound dramatically decreases free-virus infectivity. Taken together, our results show that activated TMPyP3-C17H35 effectively inhibits HSV-1 replication by several different mechanisms