The aerial parts of Alchemilla L. species (Rosaceae) are used internally as diuretic, laxative, tonic and externally for wound healing in Turkish folk medicine. Antioxidant effects of the extracts, fractions and isolated compounds from the aerial parts of A. barbatiflora Juz. were investigated with following methods: 1,1-diphenyl-2-picryl-hydrazyl (DPPH), and superoxide radical scavenging (SOD), phosphomolibdenum-reducing antioxidant power (PRAP), ferric-reducing antioxidant power (FRAP) assays. In addition, tyrosinase, a-glucosidase and acetylcholinesterase inhibition activities of samples were analyzed. The methanol extract from the aerial parts of plant was consecutively fractionated into four subextracts; n-hexane, chloroform, and remaining water extracts. Further studies were carried out on the most active water subextract and the fractions obtained from water subextract with column chromatography. Phytochemical studies on active fractions of the water subextract led to the isolation of seven metabolites including catechin (1) and a catechin dimer; procyanidin B3 (2), a flavonol glucuronide; quercetin-3-O-beta-D-glucuronic acid (miquelianin) (3) with flavonoid glycosides; quercetin-3-O-beta-D-galactoside (hyperoside) (4), quercetin-3-O-beta-D-arabinoside (guaiaverin) (5), kaempferol-3-O-beta-Dxylopyranoside (6) and kaempferol-3-O-(6''-coumaroyl-beta-D-glycoside) (tiliroside) (7). Their structures were elucidated by spectral techniques (1D and 2D NMR). The experimental data verified that procyanidin B3 displayed remarkable enzyme inhibitory activity among the whole isolated compounds.