Background: Adenovirus causes a number of diseases in human, and can cause serious infection in severely immunosuppressed individuals. Despite the seriousness of adenovirus infection, there is no definitely approved anti-adenoviral therapy. Many studies have shown that compounds derived from medicinal plants have antiviral activity. Therefore, this study evaluated in vitro anti-adenoviral activity of ethanol extract, fractions, and main phenolic compounds of pomegranate peel. Methods: The ethanol extract of pomegranate peel was prepared with maceration method and fractionated by consecutive liquid/liquid partition. The cytotoxic and anti-adenovirus activities of the extract, fractions, and main phenolic compounds (ellagic acid, punicalagin and gallic acid) were evaluated on Hep-2 cell line using MTT assay. Inhibitory effect on adsorption and post-adsorption phases of the virus replication cycle was also evaluated. Results: Pomegranate peel extract had a desirable effect against adenovirus with IC50 of 5.77 µg/mL and selectivity index of 49.9. Among the fractions and compounds, the n-butanol fraction and gallic acid had the highest anti-adenoviral activity with IC50 of 2.16 µg/mL and 4.67 µM and selectivity indices of 122.5 and 10.5, respectively. The crude extract, n-butanol fraction and gallic acid inhibited the virus replication in post-adsorption phase (p < 0.01). Conclusion: Pomegranate peel extract, especially its n-butanol fraction, could serve as a new promising anti-adenovirus agent due to high inhibitory effect against adenovirus replication. The effect of the n-butanol fraction may be related to the synergistic effect or other compounds of this fraction. Further understanding of the bioassay guided isolation of natural compounds of this fraction seems essential
