This is the first report on developing new methodologies to synthesize hydroxytelechelic natural rubber (HTNR), a bio-polyol based on natural rubber (NR), by mechanical and chemical degradation. The mechanical degradation of NR from shear during mastication leads to a lower molecular weight before oxidative cleavage by periodic acid. The effects of the molar ratio of periodic acid/isoprene unit ([H5IO6]/[isoprene unit]) on properties of HTNR were investigated. The end-functionality was determined by 1H NMR spectroscopy and the molecular weight was determined by SEC. The HTNR polyol with 3000 g/mol molecular weight and H5IO6 at 0.07 ratio were used as precursors to synthesize cationic waterborne polyurethane (cWPU). The cWPU had a well-defined size distribution and was stable for at least 3 months. The chemical structure of cWPU was confirmed by 1H NMR spectroscopy. Moreover, the hydrophilic properties and antimicrobial properties of cWPU improved when mixed with 1% solution of protonated chitosan (PCS). In addition, the effects of PCS content were studied. The investigations showed that increasing PCS gave a more hydrophilic surface with a low contact angle and a high surface free energy for the cWPU-PCS film. Furthermore, PCS at over 3%wt content inhibited E. coli growth. Hence, the new cWPU mixed with chitosan could serve as an alternative antimicrobial coating material.