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This study aimed to evaluate the antimicrobial effect of Thymoquinone (TQ) on four different oral microorganisms. Minimum Bactericidal Concentration (MBC), Minimum Inhibition Concentration (MIC), Broth microdilution, and Well diffusion tests were used to determine the optimum antimicrobial concentrations of TQ against <i>Streptococcus salivarius, Streptococcus oralis, Streptococcus mutans,</i> and <i>Staphylococcus aureus</i> over 1, 3, 6, 12 and 24 h. Chlorhexidine 0.12% was selected as a positive control. The inhibitory effect of TQ on bacterial growth was most noticeable with <i>S. salivarius,</i> while the least affected was <i>S. aureus.</i> TQ’s MBC and MIC for <i>S. oralis</i> and <i>S. aureus</i> were comparable 2 mg/mL and 3 mg/mL, respectively. <i>S. salivarius</i> was most resistant to TQ and displayed a value of 5 mg/mL and 4 mg/mL for MIC and MBC, respectively. The viable count of different strains after exposure to TQ’s MBC values was most noticeable with <i>S. aureus</i> followed by <i>S. oralis</i> and <i>S. mutans,</i> while <i>S. salivarius</i> was least affected. This study emphasized the promising antimicrobial effect of TQ against the four main oral microorganisms. It has a potential preventive effect against dental caries as well as other oral diseases.