Chlorination is one of the most widely used disinfection techniques, and the problem of "chlorine-resistant bacteria" (CRB) has attracted more attention recently. In this study, the deactivation of typical CRB in water, Mycobacterium fortuitum (M. fortuitum) and Mycobacterium mucogenicum (M. mucogenicum), by free chlorine was investigated with Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) as the reference. The chlorination effectiveness of chlorine on M. fortuitum and M. mucogenicum and the effect of chlorine concentration, pH, and humic acid were studied. It was found that M. mucogenicum was more resistant to chlorine than M. fortuitum, both of which were much more resistant than E. coli and B. subtilis. The effect of disinfectant concentration on the inactivation efficiency was positive, whereas the influence of pH and humic acid was negative. The inactivation mechanisms were explored by analyzing the bacteria morphology, the destruction of cell membrane, the cell hydrophobicity, as well as total adenosine triphosphate (ATP), superoxide dismutase (SOD) activity. The slight destruction of the cell membrane was observed after deactivation with chlorine, and high hydrophobicity of the cell membrane combined with metabolic changes might lead to the chlorine tolerance of Mycobacteria.