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The process of upcycling and incorporating food by-products into food systems as functional ingredients has become a central focus of research. Barley rootlets (BR) are a by-product of the malting and brewing industries that can be valorised using lactic acid bacteria fermentation. This research investigates the effects of the inclusion of unfermented (BR-UnF), heat-sterilised (BR-Ster), and five fermented BR ingredients (using <i>Weissella cibaria</i> MG1 (BR-MG1), <i>Leuconostoc citreum</i> TR116 (BR-TR116), <i>Lactiplantibacillus plantarum</i> FST1.7 (BR-FST1.7), <i>Lactobacillus amylovorus</i> FST2.11 (BR-FST2.11), and <i>Limosilactobacillus reuteri</i> R29 (BR-R29) in bread. The antifungal compounds in BR ingredients and the impact of BR on dough rheology, gluten development, and dough mixing properties were analysed. Additionally, their effects on the techno-functional characteristics, in vitro starch digestibility, and sensory quality of bread were determined. BR-UnF showed dough viscoelastic properties and bread quality comparable to the baker’s flour (BF). BR-MG1 inclusion ameliorated bread specific volume and reduced crumb hardness. Breads containing BR-TR116 had comparable bread quality to BF, while the inclusion of BR-R29 substantially slowed microbial spoilage. Formulations containing BR-FST2.11 and BR-FST1.7 significantly reduced the amounts of sugar released from breads during a simulated digestion and resulted in a sourdough-like flavour profile. This study highlights how BR fermentation can be tailored to achieve desired bread characteristics.