AbstractThe abundance of mycoplasma‐like microorganisms seems to be common in the intestinal tract of fish, but their occurrence in other organs can be considered pathological. Isolation of piscine mycoplasmas is essential to prove their virulence. In this study, a total of 100 specimens of nine freshwater fish species collected from natural waters and fish farms were analysed for the prevalence of mycoplasmas in different extraintestinal organs. Our results demonstrate that mycoplasmas are present in carnivorous fish (pike, wels catfish, black bullhead and pike perch) than in cyprinids more frequently. Among the surveyed organs, they colonize mainly the gills, the kidney and the livers. Although mycoplasmas could be detected in one‐fifth of samples by molecular assays, the isolation of mycoplasmas belonged to the M. sualvi cluster within the hominis group of Mycoplasmataceae, but was separate from M. mobile, has been successful only from some specimens originated from wels catfish that was feasible only by special preparation steps (prefreezing, filter sterilization and incubation conditions (long culturing period, etc.). The low‐efficiency of isolation trials indicates an extensive gap in our knowledge about piscine mycoplasmas, especially in the field of their nutritional requirements.