Pineapple by-products (peels and stems) from fruit processing industries were evaluated to understand its potential application as a functional food. Therefore, the bioactive compounds of pineapple by-products were characterized for prebiotic and antioxidant activities. A total characterization of soluble carbohydrates profile (simples and complex carbohydrates), as well as polyphenols was performed, after removal of enzymatic fraction from pineapple crude juice, allowing the decrease of proteolytic activity and improving the other biological activities. Results showed that pineapple liquid fraction, from stem and peels, can be applied as a prebiotic enhancer, promoting the growth of five probiotic microorganisms (two strains of Lactobacillus sp. and three strains of Bifidobacterium sp.), as a single carbohydrate source. Moreover, through HPLC (High Performance Liquid Chromatography) analysis, 10 polyphenols were identified in pineapple liquid fractions, with some expected differences between both evaluated by-products. Gastrointestinal tract was simulated, in a continuous mode to understand the impact of pH changes and gastrointestinal enzymes into pineapple liquid fractions. Results showed a digestion of high molecular weight polysaccharides into small molecular weight tri-, di-, and monosaccharides. There was an increase of samples antioxidant activity through the gastrointestinal stage, followed by the release of specific polyphenols, such as chlorogenic, coumaric, and ferulic acids. The prebiotic activity did not improve throughout the simulation, in fact, the prebiotic potential decreased throughout the different stages.