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Nigella sativa L. (NS) and its volatile compounds are well known for their broad spectrum of effects. This study aimed to investigate the variability of the chemical composition and the in vitro antibacterial activity of five essential oils (Eos) originated from Morocco, Saudi Arabia, Syria, India, and France. These five samples were grown under different edaphic and climatic conditions. The agar diffusion method and microdilution method in 96-well plates were used to test the sensitivity of multidrug-resistant strains clinically isolated from patients (methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii), for the determination of the minimum inhibitory concentration and bactericidal concentration. Among all the investigated Eos, the monoterpenes were highly present in the chemical composition. Moroccan, Saudi Arabian, and Syrian seeds were characterized by the presence α-phellandrene (20.03–30.54%), β-cymene (12.31–23.82 %), and 4−caranol (9.77–14.27%). The Indian seeds were rich with 4-caranol (18.81%), β-cymene (14.22%), α-phellandrene (10.58%), and β-chamigrene (9.54%), while France NS was rich with estragole (20.22%) and D-limonene (14.63%). The minimum inhibitory (MIC) and bactericidal concentration (MBC) obtained for the four Eos (with the exception of France because of the low yield) tested were ranging from 3 to 40 μl/ml. Gram-positive (+) bacteria were slightly sensitive to the Eos tested than the Gram-negative (−) bacteria. The results of this study showed that the Eos of NS seeds show interesting antibacterial activity which could be associated to the existence of different bioactive compounds. Indeed, these compounds can be used for preventive or curative purposes in the face of the noncontrolled emergence of resistance to antibiotics. [ABSTRACT FROM AUTHOR]