The aim of this work was to conduct the chemical composition of essential oil (EO) of Centaurea furfuraceafrom Algerian Sahara and investigate the antioxidant, anticholinesterase, antimicrobial and antibiofilm activities of its essential oil and methanol extract. EO of C. furfuracea was analyzed by GC and GC-MS. Sixty-nine compounds identified, representing 96.94 % of the total oil. Caryophyllene oxide (12.01 %), Z-10-pentadecen-1-ol (8.11 %), farnesyl methylester (7.79 %) were identified as the main constituents. The antioxidant activity was determined using three complementary assays, in lipid peroxidation inhibition the methanol extract showed the best activity with an IC50 = 28.73 ± 0.29 µg/mL followed by EO with an IC50 = 95.95 ± 15.20 µg/mL. While in DPPH and CUPRAC assays, EO and methanol extract indicated a less to moderate activity. The in vitro anticholinesterase activity, the methanol extract showed moderate inhibitory activities against acetylcholinesterase (IC50= 164.4 ± 5.69 µg/mL) and butyrylcholinesterase (IC50 = 82.4±1.75 µg/mL), while EO was inactive against both enzymes. Minimum inhibitory concentrations (MICs) were calculated by microtitre broth dilution method, and antibiofilm effect by microplate biofilm assay. EO inhibited the growth of all tested microorganisms, MIC values were between 6.25 and 25 µL/mL concentrations, better than methanol extract. The highest antibiofilm activity have reached to 87.90 % with methanol extract of C. furfuracea against Staphylococcus aureus ATCC 6538 Pat 50 mg/mL concentration.These results showed that C. furfuracea is a natural source of active compounds with antibiotic and antibiofilm effects against S. aureusand B. subtilis, and Bacillus cereus, respectively, and also presents antioxidant and anticholinesterase properties.