Herpesviruses require several cellular proteins for their lytic DNA replication including topoisomerase II (Topo II). Thus, Topo II could be an effective drug target against herpesviral infection. In this study, we examined several Topo II catalytic inhibitors for their potentials in blocking EBV replication and becoming efficacious antiviral agents. Topo II catalytic inhibitors in general exhibited marked inhibition of EBV lytic replication and minimal cytotoxicity. In particular, (+)-rutamarin, with the best selectivity index (SI>63) among the inhibitors tested in this study, is effective in inhibiting EBV DNA replication and virion production but shows little adverse effect on cell proliferation, suggesting its potential to become an efficacious and safe drug for the treatment of human diseases associated with EBV infection.