In vitro and in vivo tests were carried out with tiamulin and gram-negative bacterial pathogens of fish. Determination of minimum inhibitory concentration for 51 strains of seven species of gram-negative bacterial pathogens showed that only strains of Vibrio anguillarum were sensitive at 1.6–6.25 ppm, while the rest of test strains required 25– >100 ppm. Control of infection was not achieved when tiamulin was fed for 14 days at 5 or 50 mg/kg to rainbow trout (Salmo gairdneri) experimentally infected with Yersinia ruckeri.