Different extracts of the aerial parts of Biscutella raphanifolia (Brassicaceae), which has not been the subject of any study, were screened for the phytochemical content, anti-microbial, antioxidant and anti-cholinesterase activities. We used four methods to identify the antioxidant activity namely, ABTS(•+), DPPH• scavenging, CUPRAC and ferrous-ions chelating methods. Since there is a relationship between antioxidants and cholinesterase enzyme inhibitors, we used two methods to determine the in vitro anti-cholinesterase activity by the use of the basic enzymes that occur in causing Alzheimer's disease: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The extracts were also tested in vitro antimicrobial activity against various bacteria. The phytochemical study of B. raphanifolia afforded four flavonol glycosides; namely, quercetin-3-O-β-D-g1ucoside, quercetin-3-O-[β-D-glucosyl(1→2)-O-β-D-glucoside], quercetin-3-O-[β-D-glucosyl(1→3)-O-β-D-glucoside] and kaempferol-3-O-[β-D-glucosyl(1→2)-[(6'''p-coumaroyl)- β-D-glucoside], being isolated here for the first time from Biscutella raphanifolia and the genus. The ethyl acetate extract showed the highest activity in ABTS(•+), DPPH• and CUPRAC assays, while the petroleum ether extract demonstrated optimum efficiency metal chelating activity. The dicloromethane and petroleum ether extracts showed a mild inhibition against AChE and BChE. However, the petroleum ether extract showed a good antibacterial activity against the pathovars Enteropathogenic E. coli (EPEC), Enterotoxigenic E. coli (ETEC) and Enterococcus feacalis, whereas the Enterohemorrhagic E. coli (EHEC) strain was more sensitive to dichloromethane and n-butanol extracts.