The study aimed to identify and evaluate indigenous plant materials with high prebiotic potential and to formulate functional plant-based prebiotic supplements. The ability of twenty-nine fruits, vegetables, and other plant-derived materials to stimulate the growth of probiotic microorganisms was tested in vitro using Lactobacillus and Streptococcus species. Their prebiotic efficacy relative to fructooligosaccharides (FOS) as the standard was determined through in vitro analyses. Many materials exhibited significant growth promotion of >60% FOS activity and hence are highly prebiotic. There was no significant effect from the drying of materials on their prebiotic performance, which thus verifies their stability. The best plant sources were used for the formulation of three formulations, F1, F2, and F3, which were thereafter assessed in vivo using Wistar rats for six weeks. All formulations improved gut microbiota, increasing Lactobacillus counts and reducing E. coli populations. Total cholesterol, triglycerides, and blood glucose showed significant reduction, while HDL and antioxidant capacity as TEAC were increased as shown by biochemical analyses. This proves that the indigenous plant-based prebiotic formulations are effective, safe, and sustainable alternatives to synthetic prebiotics and hence have a good potential for applications in nutraceuticals and functional foods for gut and overall health improvement.