Peony as an important medicinal material is widely cultivated in China, which is one of the natural distribution centers of wild peony species. So far, however, there has not been a systematic study of the roots from China's wild peonies. In this study, the total phenolic (TPC), total flavonoid (TFC), other secondary metabolites, and microelement content, as well as the antioxidant, antibacterial, anticholinesterase, and antitumor activities of peony roots from 15 species and 2 subspecies were measured. Thirteen secondary metabolites were detected, with Paeoniflorin and Paeonol being the highest content bio-activities compounds. Additionally, the peony roots had a significant antioxidant activities and bacteriostatic effect against Gram-positive bacteria, with MIC varying from 0.063 to 1 mg/mL. P. anomala subsp. veitchii and P. lactiflora showed outstanding anticholinesterase capacities and cytotoxic activities. Taken together, the data presented here provide new insights into both the medicinal and edible potential of roots from wild peony species.