• HOS is a highly effective disinfectant for the bacterial inactivation. • ROS species verification in HOS revealed the presence of singlet oxygen. • Disparate HOS disinfection rates between S. aureus and E. coli were firstly studied. • FM, TEM, and K + leakage provide early diagnosis of altered membrane permeability. • Biophysical properties and 3D images of inactivated bacteria were firstly identified. Bacterial infection and subsequent disinfection of microorganisms are ongoing issues around the world. Bio-calcium oxide derived from heated oyster-shell (HOS) waste product has been shown to be an effective disinfectant and has the additional advantage of the marketing use of utilizing waste materials that would otherwise contribute to environmental problems; however, its mode of inactivation is unknown. In this research, fluorescence microscopy (FM), transmission electron microscopy (TEM), atomic force microscopy (AFM), and electron spin resonance (ESR) spectrometer techniques were used to characterize the mode of inactivation of S. aureus and E. coli via HOS. This is the first work to provide insight into the three-dimensional morphology and biophysical properties of the inactivated S. aureus and E. coli cells via HOS. Noteworthy, the presence of singlet oxygen in HOS suspension altered bacterial cell permeability, leading to sustained inactivation. The HOS exhibited excellent disinfection capacity and achieved a 5-log-inactivation E. coli within 60 min with a dose of 0.2 g/L, superior to other shell-derived disinfectants. Thus, HOS provides a cost-effective disinfectant for the application of controlling pathogens.