Amoebic gill disease (AGD) is a significant health issue for Atlantic salmon farmed in a marine environment. While the disease is currently managed using freshwater or hydrogen peroxide baths, there is a need to develop other treatments. The aims of this study were to examine the effect of salinity (0 ppt and 35 ppt) and temperature (3 °C and 15 °C) on attachment and survival of Neoparamoeba perurans in vitro over short exposure times (15 min and 2 h) and to assess the efficacy of reduced temperature (3 °C) as treatment for Atlantic salmon affected by AGD. In vitro freshwater 3 °C was at least as effective as freshwater 15 °C and the attachment was significantly lower after 2 h in freshwater 3 °C than freshwater 15 °C. In vivo there was no difference between the fish treated with freshwater 15 °C for 2 h or freshwater 3 °C. This study showed that despite exposure to low temperature reducing attachment of N. perurans to their substrate in vitro, 15 min cold-water bath treatment was not more effective at reducing AGD in Atlantic salmon than current commercial 2 h freshwater bath.