Mycoplasma contamination was identified in 11 cell lines derived from both coldwater and warmwater fish using DNA staining, microbiological culture and ELISA test detection methods. Acholeplasma laidlawii and Mycoplasma arginini were present as single or combined infections. Treatment of cells over three to six serial subcultivations with the addition of 0.5 μg m−1 mycoplasma removal agent (MRA) or 10 μg m−1 ciprofloxacin to culture growth medium effectively eliminated all contaminants except for one instance of M. arginini infection.