Featured Application: Bionanocellulose/κ-carrageenan composites developed within this study meet the specific tissue engineering requirements, including high tensile and compression strength, water holding capacity, and water retention ratio, suitable swelling properties as well as the positive effect on cell differentiation. As the key properties of these composites may be easily modified during their fabrication, the established procedure may lead to the production of customized scaffolds.Abstract: In this work, novel bacterial cellulose/κ-carrageenan (BNC/κ-Car) composites, being potential scaffolds for tissue engineering (TE), and outperforming the two polymers when used as scaffolds separately, were for the first time obtained using an in situ method, based on the stationary culture of bacteria Komagateibacter xylinus E25. The composites were compared with native BNC in terms of the morphology of fibers, chemical composition, crystallinity, tensile and compression strength, water holding capacity, water retention ratio and swelling properties. Murine chondrogenic ATDC5 cells were applied to assess the utility of the BNC/κ-Car composites as potential scaffolds. The impact of the composites on the cells viability, chondrogenic differentiation, and expression patterns of Col1α1, Col2α1, Runx2, and Sox9, which are indicative of ATDC5 chondrogenic differentiation, was determined. None of the composites obtained in this study caused the chondrocyte hypertrophy. All of them supported the differentiation of ATDC5 cells to more chondrogenic phenotype.