Abstract Solid fractions from pineapple stems and peels are constituted by structural carbohydrates coupled with dietary fibre, simple sugars, but also vitamins and polyphenols, which together can have potential effects on human health. The present studies report for the first time the bioavailability and bioaccessibility of pineapple by-products fractions throughout simulated gastrointestinal tract, evaluates prebiotic potential and in vitro human microbiota fermentation. The pineapple flours promoted the human faeces fermentation through growth of beneficial strains, being corroborated by the decrease of simple sugars and the production of healthy organic acids (acetic, propionic and butyric acids) - well known short chain fatty acids. On the other hand, a high phenolic compounds content was release through flours digestion, developing an antioxidant environment within human gut. Thus, was possible to conclude that pineapple flour promoted a positive modulation in the overall system, proving a synergetic interaction of dietary fibre and polyphenols upon human microbiota.