Aims: To evaluate the potential for using a novel chemiluminescence-based enzyme assay for rapid detection of enterococci in water contaminated with faecal waste. Methods and Results: The novel assay (EntLight) was based on the enzymatic hydrolysis of the chemiluminescent 1,2-dioxetane [(4-methoxy-4(3-beta-d-glucoside-4-chlorophenyl)] spiro[1,2-dioxetane-3-1,3-tricyclo[7.3.1.0(2,7)] tridec-2,7-ene] specific for beta-d-glucosidase. The specificity of the proposed EntLight assay was characterized using 26 different Enterococcus strains and 10 bacterial genera other than Enterococcus. With an analysis time of <= 8 h, the assay was found to be sensitive and specific. Validation experiments were carried out using water samples contaminated with raw municipal wastewater in comparison with qPCR and ISO standard methods. EntLight was successfully applied to detect enterococci in contaminated water within <= 8 h, and the proposed assay correlated well with both qPCR and ISO standard methods (R(2) > 0 776). Conclusions: EntLight can be applied to rapid and simple detection of viable enterococci in water contaminated with faecal matter. Significance and Impact of the Study: The novel EntLight assay and qPCR have the potential to be used as methods for early warning (1-7 h) of faecal pollutions in different water types.