Ferns are famous traditional medicinal plants and 300 fern species have been used as phytomedicines by Southwestern of China. However, there is little information on the phytochemicals and pharmacological characteristics of most fern species. The total flavonoid concentrations, antioxidant activity, anticancer activity, and acetylcholinesterase inhibition of extracts from 19 species of ferns were investigated. The total flavonoid concentrations ranged from 8.6 to 306.4 mg/g (w/w). The antioxidant activity including DPPH free radical scavenging, ABTS radical scavenging, and superoxide anion scavenging, reducing power and ferric reducing antioxidant potential (FRAP) of flavonoid extracts from these ferns showed a weak linear relationship with their total flavonoid concentrations. Selaginella frondosa with very low total flavonoid concentration (13.4 mg/g) showed highest inhibition against A549 cells and it is necessary to further investigate the anti-cancer compounds in S. frondosa. The extracts from Davallia cylindrica Ching and Stenoloma chusanum Ching significantly inhibited AChE activity, which illustrated that it is worthy of attention to investigate flavonoids from D. cylindrica and S. chusanum as AChE inhibitors.