Bacterial spores pose significant risks to human health, yet the inactivation of spores is challenging due to their unique structures and chemical compositions. This study investigated the synergistic effect between surfactants and chlorine on the inactivation kinetics of Bacillus subtilis spores. Two surfactants, cocamidopropyl betaine (CAPB) and cetyltrimethylammonium chloride (CTMA) were selected to investigate chlorine disinfection in absence and presence of surfactants. The concurrent presence of both chlorine and surfactant resulted in a moderate reduction in the lag-phases for spore inactivation and negligible increase in the second-order inactivation rate constants. In contrast, when the spores were pre-exposed to surfactants, the lag-phases decreased by about 50 % for both CAPB and CTMA, and the second-order inactivation rate constants during post-chlorination remained constant for CAPB but increased by a factor of 2.3 for CTMA, compared to the control group with phosphate buffer. This synergistic effect became more pronounced with longer surfactant pre-exposure times, reaching its maximum at 3–6 h. The observed synergistic effect suggests that surfactants can potentially enhance the permeability of the coat which is the outmost layer of B. subtilis spores and a primary barrier for chemical disinfectants. Tracing a group of B. subtilis spores sequentially treated with surfactant and chlorine by atomic force microscopy, a significant decrease in compressive stiffness of the spores was observed due to exposure to surfactants, indicating alterations in the coat by surfactants. The trend in reducing compressive stiffness aligned well with the decrease of lag-phases in inactivation kinetics. Furthermore, CTMA was found to inactivate B. subtilis spores through mechanisms different from chlorine. Chlorine primarily inactivated B. subtilis spores before damaging the inner membrane of the spores which plays a crucial role in protecting the genetic material stored in the core of the spores. In comparison, CTMA damaged 22 % of the inner membrane for an inactivation efficiency of 99 %. A synergistic effect in damaging the inner membrane was observed when applying CTMA and chlorine simultaneously instead of sequentially.