Six new prenylated acylphloroglucinols with menthane moieties, hypascyrins A-E (1-5) and ent-hyphenrone J (6), together with four known analogues, were isolated from Hypericum ascyron roots. Detailed spectroscopic data analyses resulted in the assignment of their structures. The absolute configuration of 1 was deduced by experimental and calculated ECD data, while those of 2-6 were assigned by ECD data analyses as well as chemical conversions. Hypascyrins A (1), C (3), and E (5) and ent-hyphenrone J (6) exhibited antimicrobial activity against MRSA (MIC50 values of 4.0, 8.0, 2.0, and 4.0 μM, respectively) and Bacillus subtilis (MIC values of 4.0, 4.0, 2.0, and 4.0 μM, respectively).