Chlorine dioxide is a globally recognized green and efficient disinfectant. This study aims to investigate the bactericidal mechanism of chlorine dioxide using beta-hemolyticStreptococcus(BHS) CMCC 32210 as a representative strain. BHS was exposed to chlorine dioxide, the minimum bactericidal concentration (MBC) values of chlorine dioxide against BHS were determined by the checkerboard method in preparation for subsequent tests. Cell morphology was observed using electron microscopy. Protein content leakage, adenosine triphosphatase (ATPase) activity, and lipid peroxidation were determined by kits, and DNA damage was determined using agar gel electrophoresis. The concentration of chlorine dioxide during disinfection showed a linear relationship with the concentration of BHS. Scanning electron microscopy (SEM) results showed that chlorine dioxide caused significant damage to the cell walls of BHS at a concentration of 50 mg/L, but had no significant effect onStreptococcusexposed to different exposure times. Furthermore, the extracellular protein concentration increased with increasing chlorine dioxide concentration, while the total protein content remained unchanged. The activities of Na+/K+-ATPase and Ca2+/Mg2+-ATPase decreased with increasing chlorine dioxide concentration. Chlorine dioxide treatment led to significant lipid peroxidation and DNA degradation in BHS. Leakage of intracellular components indicated that chlorine dioxide damaged the cell membrane of BHS. Chlorine dioxide exposure resulted in oxidative damage to lipids and proteins, which negatively impacted the cell wall and membrane of Streptococcus. This caused increased permeability and inactivation of key enzymes (Na+/K+-ATPase and Ca2+/Mg2+-ATPase) involved in respiratory metabolism, ultimately leading to DNA degradation and bacterial death due to either content leakage or metabolic failure.