The ability of the avian pathogen Mycoplasma gallisepticum to persist despite fluoroquinolone treatment was investigated in chickens. Groups of specific pathogen free chickens were experimentally infected with M. gallisepticum and treated with enrofloxacin at increasing concentrations up to the therapeutic dose. When M. gallisepticum could no longer be re-isolated from chickens, birds were stressed by inoculation of infectious bronchitis virus or avian pneumovirus. Although M. gallisepticum could not be cultured from tracheal swabs collected on several consecutive sampling days after the end of the enrofloxacin treatments, the infection was not eradicated. Viral infections reactivated the mycoplasma infection. Mycoplasmas were isolated from tracheal rings cultured for several days, suggesting that M. gallisepticum persisted in the trachea despite the enrofloxacin treatment. The minimal inhibitory concentration (MIC) of enrofloxacin for most of the re-isolated mycoplasmas was the same as that of the strain with which the birds were inoculated. Furthermore, no mutation could be detected in the fluoroquinolone target genes. These results suggest that M. gallisepticum can persist in chickens without development of resistance despite several treatments with enrofloxacin.