This study investigates the antibacterial activity of thymoquinone (TQ) and thymohydroquinone (THQ), two main components of black seed essential oil, against various bacteria, including Escherichia coli, Pseudomonas aeruginosa, Shigella flexneri, Salmonella Typhimurium, Salmonella Enteritidis, and Staphylococcus aureus. Both TQ and THQ exhibited antibacterial activity against both gram-positive and gram-negative bacteria, with S. aureus being highly susceptible to TQ. The MIC and MBC values for TQ and THQ against gram-negative bacteria ranged from 200 to 1600 μg/ml. The combination of TQ and THQ with antibiotics (ampicillin, cephalaxin, chloramphenicol, tetracycline, gentamicin, and ciprofloxacin) showed synergism in S. aureus, while in gram-negative bacteria, synergism, antagonism, and indifferent effects were observed in 28.9%, 23.6%, and 47.5% of the tested combinations, respectively. This study highlights the potential of TQ and THQ as antibacterial agents, particularly for S. aureus, and their enhanced activity when combined with antibiotics.This study investigates the antibacterial activity of thymoquinone (TQ) and thymohydroquinone (THQ), two main components of black seed essential oil, against various bacteria, including Escherichia coli, Pseudomonas aeruginosa, Shigella flexneri, Salmonella Typhimurium, Salmonella Enteritidis, and Staphylococcus aureus. Both TQ and THQ exhibited antibacterial activity against both gram-positive and gram-negative bacteria, with S. aureus being highly susceptible to TQ. The MIC and MBC values for TQ and THQ against gram-negative bacteria ranged from 200 to 1600 μg/ml. The combination of TQ and THQ with antibiotics (ampicillin, cephalaxin, chloramphenicol, tetracycline, gentamicin, and ciprofloxacin) showed synergism in S. aureus, while in gram-negative bacteria, synergism, antagonism, and indifferent effects were observed in 28.9%, 23.6%, and 47.5% of the tested combinations, respectively. This study highlights the potential of TQ and THQ as antibacterial agents, particularly for S. aureus, and their enhanced activity when combined with antibiotics.