Aloe vera mucilage is reported to be rich in acemannan that is a polysaccharide with a backbone of β-(1→4)-D-mannose residues acetylated at the C-2 and C-3 positions and contains some side chains of galactose and arabinose attached to the C-6 carbon. The evaluation of the prebiotic potential of Aloe vera mucilage was carried out by in vitro fermentation using intestinal microbiota from six healthy donors as the inoculum. The prebiotic activity was assessed through the quantification of short chain fatty acids (SCFA) and the evaluation of dynamic bacterial population in mixed faecal cultures by fluorescence in situ hybridization (FISH). Our findings support the possible incorporation of the Aloe vera mucilage in the development of a variety of food products known as prebiotics aimed at improving gastrointestinal health.