Drug resistant strains of herpes simplex virus-1(HSV-1) have been recently increased. Thus, new antiviral agents derived from medicinal plants which are effective against HSV-1 infections are urgently required. Therefore, this research was aimed to evaluate in vitro anti-HSV-1 activity of pomegranate (Punica granatum L.) peel extract and the investigation of antioxidant properties of these plant materials. In this research, crude ethanol extract of pomegranate peel was prepared. Anti-HSV-1 activity of the extract was evaluated on Vero cell line using MTT (3- [4,5-dimethylthiazol–2-yl]-2,5-diphenyltetrazolium bromide) assay. The 50% inhibitory concentration (IC50) and 50% Cytotoxicity Concentration (CC50) of the extract were determined using regression analysis. Its inhibitory effect on adsorption and/or post-adsorption stages of HSV-1 replication cycle was determined. To determine antioxidant activity, total phenol content, and flavonoids content of the extract, the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay; Folin-Ciocalteu method and aluminum chloride colorimetric method was used, respectively. The CC50 and IC50 of the extract were 293.5±10/1 and 37.7±6/7μg/ml, respectively. The selectivity index (SI), the ratio of CC50 and IC50, was 7.78. This extract inhibited HSV-1 replication in adsorption stage (p<0.05). The IC50 of DPPH radical was 7.7±1.21 μg/ml, compare with butylated hydroxytoluene (BHT), with IC50 of 25.41±1.89 μg/ml. The total phenol and Flavonoid contents were 282.9 mgGAE/g and 136.6mg/g, respectively. This study revealed that the pomegranate (Punica granatum L.) peel extract exhibited anti-HSV activity, probably by inhibiting the adsorption stage, with SI value of 7.7, suggesting its potential use as anti-HSV agents. Also this extract with high phytoconstituents could be a promising source of medicinally important natural compound.