The genus Centaurea is recognized in folk medicine for anti-inflammatory, anti-itch, antitussive, purgative, astringent, and tonic activities. To study the chemical determinant for antimicrobial activity essential oils (EOs), five Centaurea species were analyzed including: C. scoparia, C. calcitrapa, C. glomerata, C. lipii and C. alexandrina. Conventional hydro-distillation (HD) and microwave-assisted extraction (MAE), as new green technologies, were compared for the extraction of essential oils. GC/MS analysis identified 120 EOs including mostly terpenoid except from C. lipii and C. alexandrina in which nonterpenoids were the major constituents. Major terpenoids included spathulenol, caryophyllene oxide and alloaromadendrene oxide-2. To probe antibacterial activity, potential EO inhibitors of a bacterial type II DNA topoisomerase, DNA gyrase B were screened via an in silico molecular docking approach. Spathulenol and alloaromadendrene oxide-2 possessed the best binding affinity in the ATP- binding pocket of Gyrase B enzyme. Principal component analysis and agglomerative hierarchical clustering were used for sample classification and revealed that sesquiterpenes contributed the most for accessions classification. In vitro antimicrobial activity against Staphylococcus aureus, Escherichia coli and Aspergillus niger for all EOs were also evaluated. EOs from C. lipii, C. glomerata and C. calcitrapa exhibited significant MIC against S. aureus with an MIC value of 31.25 µg/mL.