To evaluate three previously unreported substrates for the detection of beta-glucosidase activity in clinically relevant bacteria and to compare their performance with a range of known substrates in an agar medium. The performance of 11 chromogenic beta-glucosidase substrates was compared using 109 Enterobacteriaceae strains, 40 enterococci and 20 strains of Listeria spp. Three previously unreported beta-glucosides were tested including derivatives of alizarin, 3',4'-dihydroxyflavone and 3-hydroxyflavone. These were compared with esculin and beta-glucoside derivatives of 3,4-cyclohexenoesculetin, 8-hydroxyquinoline and five indoxylics. All substrates yielded coloured precipitates upon hydrolysis in agar. Alizarin-beta-D-glucoside was the most sensitive substrate tested and detected beta-glucosidase activity in 72% of Enterobacteriaceae strains and all enterococci and Listeria spp. The two flavone derivatives showed poor sensitivity with Gram-negative bacteria but excellent sensitivity with enterococci and Listeria spp. Alizarin-beta-D-glucoside is a highly sensitive substrate for detection of bacterial beta-glucosidase and compares favourably with existing substrates. beta-glucosides of 3',4'-dihydroxyflavone and 3-hydroxyflavone are effective substrates for the detection of beta-glucosidase in enterococci and Listeria spp. The data presented allow for informed decisions to be made regarding the optimal choice of beta-glucosidase substrate for detection of pathogenic and/or indicator bacteria.