Flavobacterium psychrophilum, the etiological agent that causes bacterial coldwater disease, has been implicated in significant reductions in the numbers of salmonids reared at fish hatcheries. In this study, we performed a series of in vitro experiments to test the effectiveness of rapid temperature, pH, and osmotic pressure changes in killing three strains of the bacterium. We also evaluated the effectiveness of antibiotics (erythromycin, streptomycin, and a penicillin-streptomycin mixture) against F. psychrophilum. The bacterium tolerated temperatures of 40-50°C for up to 30 min (when acclimated to 15°C). The bacterium can survive lower temperatures for >60 min. Although temperatures ≥55°C appeared to kill F. psychrophilum on contact, we found that eyed eggs of Rainbow Trout Oncorhynchus mykiss were not able to survive short (<60-s) exposures at these temperatures. We found that rapid changes in pH (15-min exposures to pH 3.0, 4.0, 5.0, 7.0, 9.0, 10.0, and 11.0) and osmotic pressure (15-min exposures to 0, 6, 8, 10, 12% sodium chloride) were not effective at killing the bacterium. Erythromycin concentrations up to 2,000 mg/L for 15 min were also ineffective. However, streptomycin concentrations ≥5,000 mg/L killed the bacterium during a 15-min exposure. The combination of penicillin and streptomycin was also effective, killing the bacterium at doses as low as 2.5 × 10(6) IU penicillin + 2,500 mg/L streptomycin. Our trials demonstrate that elevated temperatures and the combination of penicillin and streptomycin can kill F. psychrophilum under in vitro conditions. Erythromycin and rapid changes in pH and osmotic pressure are not effective at killing the bacterium.