Six new prenylated acylphloroglucinols with menthane moieties, hypascyrins A-E (<b>1</b>-<b>5</b>) and <i>ent</i>-hyphenrone J (<b>6</b>), together with four known analogues, were isolated from <i>Hypericum ascyron</i> roots. Detailed spectroscopic data analyses resulted in the assignment of their structures. The absolute configuration of <b>1</b> was deduced by experimental and calculated ECD data, while those of <b>2</b>-<b>6</b> were assigned by ECD data analyses as well as chemical conversions. Hypascyrins A (<b>1</b>), C (<b>3</b>), and E (<b>5</b>) and <i>ent</i>-hyphenrone J (<b>6</b>) exhibited antimicrobial activity against MRSA (MIC<sub>50</sub> values of 4.0, 8.0, 2.0, and 4.0 μM, respectively) and <i>Bacillus subtilis</i> (MIC values of 4.0, 4.0, 2.0, and 4.0 μM, respectively).