Abstract: The biological effects of NaClO, H₂O₂ or UV towards gram-negative Escherichia coli and gram-positive Enterococcus hirae were investigated by using FTIR microspectroscopy. The results show significant correlations between bacterial inactivation and spectral alterations, and also distinctly different effects profiles induced by the different disinfectants and bacteria. Gram-negative bacteria were more vulnerable to disinfection than gram-positive bacteria. The most significant biological effects on membrane permeability and oxidation of proteins and lipids were observed for NaClO, followed by the oxidation effects of HO · radicals generated with photolysis-of H₂O₂, and lastly through DNA damage by UV radiation. The findings provide new insights into the biological effects of disinfection processes, and a scientific basis for optimization to ensure the water safety.