Bacterial diseases and pathogens of crayfish are common, widespread, and occasionally causing serious mortalities. In order to take rapid measures for correct treatment of crayfish diseases, the turnover time and accuracy in bacterial identification is an issue. Bacteria isolated from tissues of apparently healthy Astacus astacus and A. leptodactylus were identified by the commercial phenotypic tests (API 20E) and by the matrix assisted laser induced desorption ionization connected to the time of flight mass spectrometry (MALDI-TOF MS). For Gram-negative rods, API 20E resulted in fewer species identifications than MALDI-TOF MS (5.2% versus 52.61%). The most frequently identified genus from A. astacus and A. leptodactylus was Pseudomonas spp.: API 20E (47.82%) and MALDI-TOF MS (52.17%). Both systems identified 60.86% of total isolates identically to the genus. Hafnia alvei was the only isolate for which API 20E and MALDI-TOF MS had a concordant reading to the species. MALDI-TOF MS proved to be a powerful, low-cost, rapid tool in bacterial genus identification. This is the first report of a direct comparison between the two systems for the identification of bacteria in crayfish, and also the first report on using MALDI-TOF MS for discrimination of freshwater crayfish bacterial isolates.