<p>B vitamins are essential cofactors in cellular metabolism, influencing host physiology and microbial community dynamics. Current supplies rely on dietary intake, supplementation, or chemical synthesis, but high production cost, environmental burden, and reliance on non-renewable feedstocks underscore the need for sustainable alternatives. Microbial fermentation offers bioavailable B vitamins, offering potential gut-targeted benefits, particularly when integrated with agro-food waste valorization. This review summarizes advances in microbial production systems, including commensal and industrial strains, metabolic engineering, and co-culture approaches, alongside vitamin-specific biosynthetic pathways. Agro-food residues as low-cost renewable substrates are discussed in the context of circular bioeconomy and zero-waste principles. Special emphasis is placed on the gut microbiota, where B vitamins modulate microbial diversity, host immunity, and metabolism, and act as regulators of microbial communication, affecting quorum sensing, biofilm formation, virulence, and resistance. By interlinking agro-waste valorization, microbial biosynthesis, gut microbiota modulation, and microbial communication, this review highlights sustainable B vitamin production, identifies knowledge gaps, and outlines future directions for microbiome-targeted innovations.</p>