The virulence and increasing antibiotic resistance of certain bacterial strains creates a need for efficient and timely detection of environmental pathogens. We evaluated the kinetics of the fluorescence enhancement of cationic dyes as an assay for differentiation between bacterial specie. For several benzothiazole cationic dyes, such as 3-3'-diethylthiacyanine, we observed fluorescence enhancement in the presence of vegetative bacteria and bacterial spores. Different bacterial species manifested different rates of emission enhancement. Although staining has been a broadly used technique for the identification of bacterial species, the kinetics of the staining process has not previously been examined. We hypothesized that the kinetic parameters can be utilized as “fingerprints” for detection and identification of bacterial species. We used three different vegetative bacteria and three different bacterial spores as model organisms to test our hypothesis. Kinetic emission assays with various concentrations of bacteria and fluorophores allowed us to determine the time constants of the fluorescence enhancement and the diffusion constant of the dye into the cell wall. These time constants reflect the migration of the dye from the surrounding media to the fluorogenic microenvironment within the bacterial cell wall. Development of this assay breaks a century old tradition of identifying bacteria using differential staining techniques and in addition it allows us to move us away from obtaining a strictly Boolean outcomes.