Selenium (Se) is a necessary essential component that has antioxidant, immune-modulating, and anticancer activities. Emerging research highlights the role of gut microbiota in colorectal cancer, with probiotics like Lactobacillus and Bifidobacterium potentially enhancing treatment efficacy, reducing inflammation, and supporting gut homeostasis. Probiotics, particularly Bacillus subtilis subsp. stercoris, play a vital role in gut microbiota regulation, improving gastrointestinal health and nutrient bioavailability. In this study, Bacillus subtilis subsp. stercoris was isolated from fermented sorghum (Koozh), a traditional Tamilian fermented food, and enriched with sodium selenite to enhance its functional properties. The selenium enrichment process resulted in a distinct orange pigmentation, indicating the biotransformation of inorganic selenium into bioavailable organic selenium compounds. The enriched strain was characterized for probiotic potential, including gastric fluid and bile salt tolerance, carbohydrate fermentation, and hydrophobicity. To evaluate its anticancer effects, an MTT assay was performed on Caco-2 colorectal cancer cells, revealing dose-dependent cytotoxicity with an IC50 of 81.517 µg/mL. Further AO/ETBR staining confirmed apoptosis induction in treated cancer cells, while maintaining good viability in non-cancerous cells. Selenium-enriched probiotics, particularly Bacillus subtilis subsp. stercoris, show promise as functional food supplements with potential anticancer and gut health benefits, highlighting their therapeutic potential as nutraceuticals for probiotic formulations, functional foods, and colorectal cancer prevention.