Inhibition of tumor promoter-induced Epstein-Barr virus (EBV) activation was screened using tissue culture and thallus extracts of lichens. Usnea longissima ACH. thallus and Cetraria ornata MULL. ARG. tissue culture showed strong inhibitory activity. We identified (+)-usnic acid (1), barbatic acid (2), diffractaic acid (3), 4-O-demethylbarbatic acid (4), and evernic acid (5) as inhibitors of EBV activation from the U. longissima thallus. Of these compounds, (+)-usnic acid exhibited the highest inhibitory activity (IC50 = 1.0 microM).