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Steady-state and time-resolved fluorescence spectroscopy were measured for five species of bacteria (Bacillus subtilis, Staphylococcusaureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa) subjected to three bactericidal agents, formaldehyde, sodium hypochlorite (bleach), and hydrogen peroxide. For all species, the fluorescence was dominated by tryptophan emission with a fluorescence lifetime that can be described by a bi-exponential decay profile. Application of bleach resulted in an almost total loss of scattering and fluorescence, which indicated that total destruction of proteins and amino acids may have occurred. Hydrogen peroxide decreased the fluorescence intensity and shifted λmax to shorter wavelengths, except in S. aureus, which is resistant to oxidizing agents. The formaldehyde shifted λmax to shorter wavelengths for B. subtilis, S. aureus, E. faecalis, and E. coli. The formaldehyde shortened the lifetime of the slow component and increased the amplitude of the fast component relative to the slow component. This study demonstrates that fluorescence spectroscopy offers a method to evaluate the potential for killing bacteria and decontaminating areas by disinfecting agents.