Triticum dicoccum (Farro) an ancient wheat species has recently gained attention for its exceptional health benefits. However, research on its antibacterial and anti-biofilm properties remains limited. Additionally, a growing trend has been observed in releasing enriched or fortified whey beverages to enhance their functionality. Therefore, this study aimed to investigate the inhibitory effects of whey beverages supplemented with enzyme-rich fermented farro (WF) on Salmonella Typhimurium biofilm formation and explore the underlying mechanisms. Treatment with WF significantly reduced biofilm formation and viability of S. Typhimurium. Moreover, WF decreased the bacterial adhesion to and invasion of human intestinal epithelial cells. WF also inhibited gene expression associated with motility and initial adhesion in S. Typhimurium, as well as genes involved in quorum sensing (QS), in a concentration-dependent manner. Furthermore, WF suppressed the production of the QS signaling molecule autoinducer-2 in a similar concentration-dependent manner. Consequently, our findings indicate that the addition of enzyme-rich fermented farro to whey beverage enhances anti-biofilm activity, which is probably attributed to its antimicrobial effects, inhibition of initial adhesion, and QS reduction. These findings offer a promising basis for developing fortified dairy beverages that can enhance food safety and promote human health.