ABSTRACT The effects of cellulase treatment on the prebiotic activity and structural properties of by-products from guava (Psidium guajava L.) purée processing was investigated focusing on Refiner (the seed-rich fraction), siever (the peel-rich fraction), and decanter (the pulp-rich fraction). After cellulase treatment, each by-product was separated into ethanolic extract (EEC) and alcohol insoluble fiber (AIF). Cellulase treatment led to the dissolution of the insoluble fiber in all the by-products. Subsequently, the molecular weight distribution and prebiotic activity of the EEC were determined. All cellulase-treated by-products showed a shift from high to low in molecular weight distribution, with a higher prebiotic activity score. Cellulase treatment also affected the microstructure, crystallinity index, and chemical bonds in AIF as observed through Scanning Electron Microscope, X-ray crystallography analysis, and Fourier-transform Infrared spectra analysis. The structural lattice changes manifested as an improved capacity in binding bile acids. Cellulase treatment is a feasible method to unleash the recalcitrant nature of guava by-products.