Traditional microbiological techniques are used to provide reliable data on the rate and extent of kill for a range of biocides. However, such techniques provide very limited data regarding the initial rate of kill of fast-acting biocides over very short time domains. This study describes the application of a recombinant strain of Escherichia coli expressing the Photorhabdus luminescens lux operon as a whole-cell biosensor. Light emission is linked directly to bacterial metabolism; therefore, by monitoring light output, the impact of fast-acting biocides can be assessed. Electrochemically activated solutions (ECASs), bleach, Virkon, and ethanol were assessed at three concentrations (1%, 10%, 80%) in the presence of organic soiling. Over a 2-s time course, 80% ECAS produced the greatest reduction in light output in the absence of organic load but was strongly inhibited by its presence. Eighty percent ethanol outperformed all tested biocides in the presence of organic soil. Bleach and Virkon produced similar reductions in bioluminescence at matched concentrations within the time course of the assay. It was also demonstrated that the assay can be used to rapidly assess the impact of organic soiling. The use of bioluminescent bacteria as whole-cell bioreporters allows assessment of the relative efficacies of fast-acting biocides within milliseconds of application. The assay can be used to investigate activity over short or extended time domains to confirm complete metabolic inhibition of the bioreporter. Moreover, the assay may enable further elucidation of their mechanism of action by allowing the investigation of activity over time domains precluded by traditional microbiology.