Bacterial cellulose (BC) is extensively used in many fields due to its excellent physical properties; however, its high production cost and low output remain a challenge. The production of conventional yellow wine typically results in the generation of a substantial quantity of rice soaking wastewater (RSW). In this study, BC producing strain was isolated and identified from Kombucha. BC was produced directly using unsterilized RSW as the medium. High-yield BC was obtained by co-fermentation of isolated Novacetimonas hansenii S-08 with natural fermentation flora from RSW. The culture conditions were optimized by combining RSW medium and seed liquid of HS medium at a ratio of 5:1, using unsterilized RSW as a medium containing 40 g/L fructose corn syrup and 2 % ethanol. The results of FTIR, XRD, TGA, SEM analysis, and mechanical properties showed that the physical properties of BC-RSW were comparable with ordinary BC. The BC-RSW had strong inhibitory effects on both Gram-positive and Gram-negative bacteria. In conclusion, the wastewater produced by yellow wine enterprises could be directly used as an alternative medium for the sustainable biosynthesis of antibacterial BC in situ.