The purpose of this investigation was to develop a new method for extracting polysaccharides from Aloe vera, considering their inhibitory effectiveness.Acemannan, the major functional Aloe vera gel polysaccharide, was gently deacetylated, in presence of sodium borohydride, in order to obtain water-soluble polysaccharides. Varying deacetylation time leads to different deacetylation degrees, which were determined via potentiometric titration.Physical and structural properties of different polymers were assessed. Fourier Transform Infrared spectroscopy confirmed acemannan deacetylation. The molecular weights were examined via Size Exclusion Chromatography analysis. Neutral sugars and uronic acid contents were also determined.Furthermore, the antibacterial activity was evaluated using counting assays. AVB5, the less deacetylated polymer, exhibited the highest antibacterial activity against four different bacteria stains, with an inhibition greater than 98%.Moreover, the strongest inhibition of E. Coli and E. faecalis biofilms formation was observed with AVB5 through BIC50 ≤ 7.6 ± 0.7 and BIC90 ≤ 68.2 ± 4.5. It was also found to be the most efficient to eradicate the biomass, via the assessment of BEC50 ≤ 10.9 ± 0.6 and BEC90 ≤ 75.5 ± 4.Collectively, deacetylation treatment of Aloe vera acemannan reduces considerably antibacterial and antibiofilm potential.