The aim of this study is to evaluate in vitro anti-adenovirus property, antioxidant potential, and total phenolic content of dried flowers buds of Syzygium aromaticum crude extract. The crud extract was prepared and its anti-adenovirus activity was investigated on HEp2 cell line using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, Folin-Ciocalteu method and aluminum chloride colorimetric method was used to determine antioxidant activity, total phenol content, flavonoids and flavonols content of the extract, respectively. Based on results, the 50% cytotoxicity concentration (CC 50 ) and the 50% inhibitory concentration (IC 50 ) of the extract were 97.66±11.4 and 4.73±1.6 µg/ml, respectively, with the selectivity index (SI) of 20.64. The crud extract inhibited adenovirus replication in post-adsorption step (p<0.05). The extract showed remarkable scavenging activity with IC 50 values of 10.05±1.93 μg/ml. Total phenolic, flavonoid and flavonol content of the crude extract was 255.8±3.95 mg GAE/g, 63.9±2.35 mg RUT/g and 62±2.35 mg RUT/g, respectively. The results of the present study indicated that S. aromaticum crude extract exhibited anti-adenovirus activity with inhibitory effect on adenovirus replication could be considered as a potential anti-adenovirus agents.