Thymoquinone (TQ) and thymohydroquinone (THQ), major components of Nigella sativa L. essential oil, were tested for antibacterial activity against various bacteria, including gram-positive (S. aureus) and gram-negative (E. coli, P. aeruginosa, S. flexneri, S. enteritidis) strains. Both compounds showed antibacterial activity, with S. aureus being highly susceptible to TQ, requiring only 3 and 6 µg/ml to inhibit and kill the bacteria, respectively. THQ required 400 and 800 µg/ml for the same effect, 100 times higher than TQ. Gram-negative bacteria were less susceptible, with MIC and MBC ranging from 200 to 1600 µg/ml. When combined with antibiotics (ampicillin, cephalexin, chloramphenicol, tetracycline, gentamicin, ciprofloxacin), TQ and THQ exhibited synergistic effects against S. aureus. In gram-negative bacteria, synergism, antagonism, and indifferent effects were observed in 28.9%, 23.6%, and 47.5% of combinations, respectively. The study demonstrated that TQ and THQ have antibacterial activity, which can be enhanced by antibiotics, especially against S. aureus. Nigella sativa oil has been traditionally used for various health purposes and may have potential as a food preservative or in combination with antibiotics for treating infections. The findings suggest that TQ and THQ could be used as antibacterial agents, either alone or in combination with antibiotics.Thymoquinone (TQ) and thymohydroquinone (THQ), major components of Nigella sativa L. essential oil, were tested for antibacterial activity against various bacteria, including gram-positive (S. aureus) and gram-negative (E. coli, P. aeruginosa, S. flexneri, S. enteritidis) strains. Both compounds showed antibacterial activity, with S. aureus being highly susceptible to TQ, requiring only 3 and 6 µg/ml to inhibit and kill the bacteria, respectively. THQ required 400 and 800 µg/ml for the same effect, 100 times higher than TQ. Gram-negative bacteria were less susceptible, with MIC and MBC ranging from 200 to 1600 µg/ml. When combined with antibiotics (ampicillin, cephalexin, chloramphenicol, tetracycline, gentamicin, ciprofloxacin), TQ and THQ exhibited synergistic effects against S. aureus. In gram-negative bacteria, synergism, antagonism, and indifferent effects were observed in 28.9%, 23.6%, and 47.5% of combinations, respectively. The study demonstrated that TQ and THQ have antibacterial activity, which can be enhanced by antibiotics, especially against S. aureus. Nigella sativa oil has been traditionally used for various health purposes and may have potential as a food preservative or in combination with antibiotics for treating infections. The findings suggest that TQ and THQ could be used as antibacterial agents, either alone or in combination with antibiotics.