Evaluation of antibacterial substantivity of Nigella sativa (0.1%) mouthwash. Introduction: Nigella sativa (Black seed) is a miraculous herb with a rich historical and religious background. It is known to have medicinal properties and the key ingredient being Thymoquinone which is known to have anti-inflammatory and anti-oxidative properties that has been found to have an important role in prevention of periodontal disease. Objective: To assess and compare the antibacterial substantivity of Nigella sativa mouthwash (0.1%) with chlorhexidine gluconate mouthwash (0.2%). Methodology: Twenty human extracted teeth were sectioned to obtain only crowns. These sectioned crowns were randomly divided into two groups to evaluate the substantivity by immersing in Nigella sativa mouthwash (0.1%) and Chlorhexidine gluconate mouthwash (0.2%) for one minute, followed by immersion in 10 ml of artificial saliva. After an interval of eight and twelve hours, an aliquot was taken from the tubes and analysed by ultraviolet spectrophotometer at 260nm. Results: The mean substantivity value of Nigella sativa (0.1%) group was relatively higher as compared to chlorhexidine gluconate (0.2%) group at eight hours (P=0.08) and twelve hours time interval (P=0.19). Conclusion: The mean substantivity value of NS was found to be relatively higher as compared to chlorhexidine gluconate at eight and twelve hours time interval. Therefore NS mouthwash (0.1%) can be used twice daily for its maximum antibacterial effect.