The total phenolic, flavonoid and tannin contents of Scabiosa arenaria Forssk roots were determined by colorimetry. The in vitro antioxidant activity was characterized by varied assays like 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), reducing power and β-carotene bleaching inhibition activity. The anti-acetylcholinesterase activity was also evaluated. Total phenolics and flavonoids contents were highest in the butanol fraction with 82.36mg gallic acid equivalents/g and 5.86mg quercetin/g of dry weight, respectively. This fraction exhibited an interesting antioxidant activity, mainly with the β-carotene bleaching inhibition test (IC50=0.018±0.009mg/mL). The same fraction showed the best anti-acetylcholinesterase activity (IC50=0.02±0.003mg/mL).The major phenolic compounds of the butanol extract were identified by RP-HPLC (reverse phase-high-performance liquid chromatography). A total of nine compounds were characterized, including p-coumaric acid, N-propyl gallate, myricetin and methyl-4-hydroxybenzoate. The active compounds identified and quantified in this fraction were mainly responsible for these in vitro biological activities.