Pyrazines are organic compounds with a varied, intense aroma of roasted nuts, occasionally with hints of baked potatoes, almonds, and others. As a result, they are used in the food industry as food flavorings. Biosynthesis of pyrazines using microorganisms in environmentally friendly conditions is an alternative to chemical synthesis. However, screening is required to isolate efficient producer strains for efficient biosynthesis of this compound. The study’s goal was to assess the ability of Bacillus subtilis cultures isolated from natto (fermented soybeans) to biosynthesize a broad range of alkylpyrazines. B. subtilis isolated cultures were found to be capable of producing 2-methylpyrazine, 2,3-dimethylpyrazine, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2,3,5-trimethylpyrazine, and 2,3,5,6-tetramethylpyrazine. As a result of the screening, two cultures of B. subtilis capable of producing alkylpyrazines were isolated. At a total concentration of 3261 µg/L, the BcP4 strain primarily produced 2-methylpyrazine (690 µg/L), 2,3-dimethylpyrazine (680 µg/L), and 2,6-dimethylpyrazine (1891 µg/L). At a total concentration of 558 mg/L, the BcP21 strain produced 2,5-dimethylpyrazine (4.5 mg/L), 2,3,5-trimethylpyrazine (52.6 mg/L), and 2,3,5,6-tetramethylpyrazine (501.1 mg/L). The results show that different B. subtilis strains are predisposed to produce different alkylpyrazines.