In this study, we investigated the partition and separation of subfractions from methanol extracts of Usnea barbata (MEUB) using solvents of different polarities, trapping the active compounds into a single subfraction, referred to as UBM6, and focusing on its antioral cancer activities. The cell proliferation and UBM6's anticancer mechanisms were assessed using ATP and flow cytometry assays on drug-treated oral cancer and normal cells. Regarding IC50, UBM6 was selected to explore MEUB's antioral cancer effects and mechanisms because it exhibited the greatest antiproliferative effect on oral cancer cells when compared to the other 13 subfractions. Without having a detrimental impact on normal oral cells (S–G), UBM6 showed selective antiproliferative effects against oral cancer cells (Ca9-22 and CAL 27), according to ATP assays. Mechanistically, UBM6 distorted cell cycle progression, stimulated cellular and mitochondrial reactive oxygen species (ROS), depleted the mitochondrial membrane potential and cellular glutathione content, promoted apoptosis and signaling, and enhanced DNA damage (γH2AX and 8-hydroxy-2-deoxyguanosine) to a greater extent in oral cancer cells than in normal control cells. N-acetylcysteine attenuated these antioral cancer effects and mechanisms. In conclusion, UBM6 is safe for normal cells and leads to oxidative stress-dependent antiproliferation, apoptosis, and oxidative DNA damage in oral cancer cells.