The wide application of chlorine disinfectant for drinking water treatment has led to the appearance of chlorine-resistant bacteria, which pose a severe threat to public health. This study was performed to explore the physiological-biochemical characteristics and environmental influence (pH, temperature, and turbidity)of seven strains of chlorine-resistant bacteria isolated from drinking water. Ozone disinfection was used to investigate the inactivation effect of bacteria and spores. The DNA concentration and cell surface structure variations of typical chlorine-resistant spores (Bacillus cereus spores)were also analysed by real-time qPCR, flow cytometry, and scanning electron microscopy to determine their inactivation mechanisms. The ozone resistance of bacteria (Aeromonas jandaei < Vogesella perlucida < Pelomonas < Bacillus cereus < Aeromonas sobria)was lower than that of spores (Bacillus alvei < Lysinibacillus fusiformis < Bacillus cereus)at an ozone concentration of 1.5 mg/L. More than 99.9% of Bacillus cereus spores were inactivated by increasing ozone concentration and treatment duration. Moreover, the DNA content of Bacillus cereus spores decreased sharply, but approximately 1/4 of the target genes remained. The spore structure exhibited shrinkage and folding after ozone treatment. Both cell structures and gene fragments were damaged by ozone disinfection. These results showed that ozone disinfection is a promising method for inactivating chlorine-resistant bacteria and spores in drinking water.