A substrate which can be used for the direct identification of pathogenic bacteria belonging to the genus Listeria by detecting an esterase activity other than Phosphatidyl Inositol-specific Phospholipase C (PI-PLC), an esterase which is specific to the species Listeria monocytogenes. The use of two substrates, one as described above and the other specific for all or some members of the genus Listeria, and a reaction medium containing such a substrate or such a combination of substrates are disclosed. An identification method which exploits such culture media is also disclosed. The invention is particularly applicable in the field of diagnosis.