Helicobacter pylori become a major cause of serious health problems and associated with an immense prevalence worldwide responsible for chronic gastritis and peptic ulcer. Therefore the effectiveness of Nigella sativa seeds against H. pylori was evaluated due to its broad range of pharmacological and antibacterial potential against pathogenic bacteria. The maceration method was followed to obtain crude seed extract (22.6 %) using methanol (90 %). This extract was further partitioned into subsequent fractions by following solvent-solvent extraction method using organic solvents according to their increasing polarity order. The percent yield of the resultant fractions were hexane (29.68 %), dichloromethane (15.6 %), ethyl acetate (1.56 %), butanol (18.75 %) and aqueous (34.37 %). The hexane fraction contained the highest value of total phenolic content (43.23 mg GAE/mL) followed by crude extract (40.88 mg GAE /mL). The total flavonoid content present in aqueous fraction (0.62 mg QE/mL) found higher as compared to the ethyl acetate fraction (0.437 mg QE/mL). The ethyl acetate fraction showed the highest percentage of scavenging activity (63.1 %) at 1000 ppm as compared to the other varying concentrations of studied fractions. Thymoquinone (TQ) was estimated and isolated by HPLC. The crude extract showed significantly higher amount of TQ (42.5 %) followed by present in hexane fraction (27.6 %). The thymoquinone was not detected in butanol fraction. The GC-MS analysis of fractions identified a total number of twenty one bioactive compounds including TQ. The FTIR analysis showed the presence of certain functional groups to the bioactive compounds in each fraction, which highlighted the importance of hexane and ethyl acetate fraction for containing prime antioxidant potential. The H. pylori bacterium was isolated and identified from the biopsy samples of infected patients (H. pylori positive). The in-vitro antibacterial activity was carried out against H. pylori, Klebsiella pneumoniae and Staphylococcus aureus. All the fractions and pure TQ was found active against H. pylori with zone of inhibition diameter (mm) ranged between 5.3 to 16.7 mm with minimum inhibitory concentration (MIC) varied in a range from 256 to 2048 μg/mL. Amongst the studied fraction the ethyl acetate fraction and TQ (256 μg/mL) demonstrated an equal highest inhibitory activity against the tested organisms followed by aqueous fraction (512 μg/mL). The butanol fraction with MIC (2048 μg/mL) remain inactive against all tested isolates. The radiolabelling of TQ (100 %) with technetium (Tc-99m) was achieved by following biodistribution and scintigraphic studies. The in-vitro and in-vivo assays were performed to assess the effectiveness of newly synthesized 99mTc- thymoquinone compound in an induced H. pylori infected mice model. The bioactivity of 99mTc-TQ was tested in an internalization assay demonstrated a promising interaction with muscle cancer cell line. Later on an effective dose of N. sativa seed was also determined through an in-vivo study designed for the treatment of H. pylori positive patients. Among the suggested trial dose rates the amount of 2.5 gram of N. sativa seeds per day had shown a promising inhibitory potential against patient’s recovery (80 %) from H. pylori. Current results provided prospects for the use of N. sativa seeds as a newly dietary supplement. TQ was proved to possess potential against H. pylori and contributed as a natural antimicrobial compound will possibly be used as a substitute for antibiotics.