Aims: To synthesize novel substrates for the detection of β-ribosidase and assess their potential for the differentiation of Gram-negative bacteria. Methods and Results: Two novel chromogenic substrates, 3′,4′-dihydroxyflavone-4′-β-D-ribofuranoside (DHF-riboside) and 5-bromo-4-chloro-3-indolyl-β-D-ribofuranoside (X-riboside) were evaluated along with a known fluorogenic substrate, 4-methylumbelliferyl-β-D-ribofuranoside (4MU-riboside). A total of 543 Gram-negative bacilli were cultured on media containing either DHF-riboside or X-riboside. Hydrolysis of DHF-riboside or X-riboside resulted in the formation of clearly distinguishable black or blue-green colonies, respectively. Hydrolysis of 4MU-riboside was evaluated in a liquid medium in microtiter trays and yielded blue fluorescence on hydrolysis which was measured using fluorimetry. β-Ribosidase activity was widespread with 75% of strains, including 85·6% of Enterobacteriaceae, showing activity with at least one substrate. Genera that demonstrated β-ribosidase activity included Aeromonas, Citrobacter, Enterobacter, Escherichia, Hafnia, Klebsiella, Morganella, Providencia, Pseudomonas, Salmonella and Shigella. In contrast, strains of Proteus spp., Acinetobacter spp., Yersinia enterocolitica, Vibrio cholerae and Vibrio parahaemolyticus generally failed to demonstrate β-ribosidase activity. Conclusions: The novel substrates DHF-riboside and X-riboside are effective for the detection of β-ribosidase in agar-based media and may be useful for the differentiation and identification of Gram-negative bacteria. Significance and Impact of the Study: This is the first report describing the application and utility of chromogenic substrates for β-ribosidase. These substrates could be applied in chromogenic media for differentiation of Gram-negative bacteria.