The study shows the influence of the origin of plant material and biological source on the in vitro antioxidant (neutralization of DPPH and OH radical, nitric oxide, and inhibition of lipid peroxidation) and anticholinesterase activity of chemically characterized and quantified ethanol extracts of ten St. John's wort samples. The investigated samples were: five Hypericum perforatum species representatives collected at different localities, one commercial sample of Hyperici herba purchased at a local market and four Hypericum species autochtonous to the Balkan Peninsula (H. maculatum subsp. immaculatum, H. olympicum, H. richeri subsp. grisebachii and H. barbatum). All the examined extracts exhibited notable antioxidant potential, but in most of the cases indigenous Hypericum species expressed stronger effects compared to the original source of the drug, H. perforatum. The changes in the content of phenolic compounds, especially flavonoids, hyperforin and hypericin, related to the source of the drug affected the investigated activities. Since all of the investigated species have shown prominent inhibition of acetylcholinesterase in vitro activity, they could be further investigated as potential substances in preventing of Alzheimer's disease.