Aims: Enzyme substrates based on 4-methylumbelliferone are widely used for the detection of Escherichia coli and enterococci in water, by detection of b-glucuronidase and b-glucosidase activity respectively. This study aimed to synthesize and evaluate novel umbelliferone-based substrates with improved sensitivity for these two enzymes. Methods and Results: A novel b-glucuronide derivative based on 6-chloro-4-methylumbelliferone (CMUG) was synthesized and compared with 4-methylumbelliferyl-b-d-glucuronide (MUG) using 42 strains of E. coli in a modified membrane lauryl sulfate broth. Over 7 h of incubation, the fluorescence generated from the hydrolysis of CMUG by E. coli was over twice that from MUG, and all of the 38 glucuronidase-positive strains generated a higher fluorescence with CMUG compared with MUG. Neither substrate caused inhibition of bacterial growth in any of the tested strains. Four b-glucosidase substrates were also synthesized and evaluated in comparison with 4-methylumbelliferyl-b-dglucoside(MU-GLU) using 42 strains of enterococci in glucose azide broth. The four substrates comprised b-glucoside derivatives of umbelliferone-3-carboxylicacid and its methyl, ethyl and benzyl esters. Glucosides of the methyl,ethyl and benzyl esters of umbelliferone-3-carboxylic acid, were found to be superior to MU-GLU for the detection of enterococci, especially after 18 h of incubation, while umbelliferone-3-carboxylic acid-b-d-glucoside was inferior. However, the variability in detectable b-glucosidase activity among the different strains of enterococci in short-term assays using the three carboxylate esters(7 h incubation) may compromise their use for rapid detection and enumerationof these faecal indicator bacteria. Conclusions: The b-glucuronidase substrate CMUG appears to be a more promising detection system than the various b-glucosidase substrates tested. Significance and Impact of the Study: The novel substrate CMUG showed enhanced sensitivity for the detection of b-glucuronidase-producing bacteriasuch as E. coli, with a clear potential for application in rapid assays for the detection of this indicator organism in natural water and other environmental samples.