Background and ObjectivesTraditional Korean rice wine (makgeolli), contains various microorganisms, including lactic acid bacteria (LAB). This study investigated the utilization of biji, a by‐product of soy processing, as a substitute for rice in makgeolli fermentation. We evaluated the functional properties of biji‐makgeolli and examined the probiotic properties of LAB isolated from it.FindingsBiji‐makgeolli exhibited lower sugar and alcohol contents and higher pH and acidity than 100% rice makgeolli (control). Moreover, biji‐makgeolli displayed higher total viable cells (5.54‐6.32 log CFU/g), yeast (4.23‐6.59 log CFU/g), and LAB (5.44‐6.48 log CFU/g) than control (5.26, 2.59, and 5.23 log CFU/g), which increased with biji addition. Furthermore, biji‐makgeolli showed higher total phenolic content, antioxidant capacity, and α‐glucosidase inhibitory activity, increasing with biji addition. Three LAB strains were isolated from makgeolli, identified as Pediococcus pentosaceus (P. pentosaceus)‐KX886792 in control (CW) and P. pentosaceus‐KX886790 (RW) and Lactobacillus coryniformis (L. coryniformis) (RM) in 50% biji replaced makgeolli. All groups (CW, RW, RM) survived exposure to pH 3 for 3 h and 0.3% bile salts for 24 h and exhibited the ability to adhere to HT‐29 cells. RW (20.7%) and RM (27.5%) showed antioxidant effects, having higher ABTS+ values than CW (7.5%). The expression of TNF‐α and IL‐8 was significantly suppressed in RW and RM compared with that in CW. RW and RM showed antimicrobial activity against E. coli. Additionally, RM was active against Staphylococcus aureus and exhibited a cholesterol‐lowering effect.ConclusionsBiji enhances the antioxidant capacity of makgeolli, and LAB isolated from biji‐makgeolli exhibit potential probiotic properties.Significance and NoveltyBiji is a potential additive for producing plant‐derived LAB in makgeolli and developing functional foods.This article is protected by copyright. All rights reserved.