This study evaluated the antibacterial spectrum and safety of a chemically modified biopolymeric flocculant (TMB) against waterborne pathogens. The biopolymer previously characterized as polysaccharide with flocculating activity is produced extracellularly by the bacterium Klebsiella terrigena. The amino sugars on the polymer were chemically modified by quaternization, which resulted in N,N,N trimethyl biopolymer (TMB). Quaternization was effective in imparting biocidal activity to TMB against five selected waterborne pathogens, namely, Aeromonas hydrophila, Yersinia enterocolitica, Salmonella typhimurium, Listeria monocytogenes and Escherichia coli O157:H7. 99.999% inactivation was achieved with S. typhimurium at a dose of 60 μg ml−1 of TMB within 60 min at the ambient temperature, followed by other pathogens. Haemotological, histopathological and general examinations indicated no adverse effects in Swiss albino mice fed with the quaternized biopolymer (120 mg kg−1 body weight−1 day−1) over a period of 30 days. These results suggested that TMB was tolerated well without any signs of toxicity and may have potential application as a safe, antimicrobial bioflocculant for both removing and inactivating waterborne pathogens.