Abstract <p>This study is aimed to evaluate the prebiotic potential of plant polysaccharides of various structural types and origins, including acacia gum arabinogalactan, beech wood xylan, apple pectin enriched in homogalacturonan, flaxseed mucilage (containing arabinoxylan, and rhamnogalacturonan I), and chicory inulin. All carbohydrate samples varied in their monosaccharide composition and molecular weight distribution. The prebiotic activity of the polysaccharides was assessed using <i>in vitro</i> models under anaerobic conditions with the <i>Bifidobacterium bifidum</i> 791 and <i>Lactobacillus acidophilus</i> n.v. Ep 317/402. It was found that adding various polysaccharides to the nutrient media intensified lactic acid production, increased the biomass yield of bifidobacteria and lactobacilli, and influenced the rate and extent of substrate assimilation. The lower molecular weight inulin (4.2 kDa) and arabinogalactan (115 kDa) supported higher biomass yields in both strains. The viable cell count of <i>Bifidobacterium bifidum</i> 791 (colony-forming units per mL) increased by 3.5, 12.3, 110.5, 128.1, and 215.8% compared to the control upon the addition of beech wood xylan, flaxseed mucilage polysaccharides, apple pectin, inulin, and acacia arabinogalactan, respectively. For <i>L. acidophilus</i> n.v. Ep 317/402, the addition of the same polysaccharides increased the biomass yield by 1.5, 7.7, 64.6, 69.2, and 207.7%, respectively, versus the control. Flaxseed mucilage, composed of rhamnogalacturonan I (3400 kDa) and arabinoxylan (1330 kDa), supported a high biomass yield in both cultures. Based on this research, it can be concluded that flaxseed mucilage promoted probiotic growth in a manner similar to, and often more effectively than, the established prebiotic inulin. Due to the availability of flaxseeds, simple mucilage extraction process, and the limited existing research, flaxseed polysaccharide complex emerges as a promising candidate for further development as a prebiotic supplement.</p>