Bacillus subtilis plays a key role in traditional plant fermentations in Africa and Asia. Here, we characterized B. subtilis strains isolated from iru, a fermented condiment from Nigeria, and evaluated their potential as riboflavin- and folate-producers. Eighty-six Bacillus spp. were isolated. The isolates were phenotypically identified, assessed for enzymatic activity (proteases, amylases), and tested for safety (hemolysis, gelatinase, and biogenic amine production). Selected isolates were screened for riboflavin and folate production in chemically defined media. Thirty-four strains were identified as B. subtilis with riboflavin production ranging from 0.006 to 0.039 mg L-1, while nine produced folates between 0.300 and 1.940 mg L-1. These strains were exposed to selective pressure of toxic analogues of riboflavin and folate, the best strain show increases from 0.013 to 0.020 mg L-1 for riboflavin and 1.665 to 3.715 mg L-1 for folate. The most promising candidates were employed for lab-scale iru fermentation, which results in vitamin levels enhancement by 38% for riboflavin (0.036 to 0.050 mg g-1 of iru) and 35% for folate (0.035 to 0.047 mg g-1 of iru) signifying that 10 g of iru fermented with these strains can provide 25% of daily riboflavin intake and 100% of folate intake. These results highlight the potential of selected B. subtilis strains as functional starter cultures for the dual purpose of iru fermentation and vitamin biofortification. Such strain selection and optimization represent a promising biotechnological strategy to enhance the micronutrient profile of traditional fermented foods while preserving their sensory and cultural value.