Freshwater biota experience physiological challenges in regions affected by salinization, but often the effects on particular species are poorly understood. Freshwater turtles are of particular concern as they appear to have limited ability to cope with environmental conditions that are hyperosmotic to their body fluids. Here, we determined the physiological responses of two Australian freshwater chelid turtles, Emydura macquarii and Chelodina expansa, exposed to freshwater (0%) and brackish water (15%, representing a hyperosmotic environment). Brackish water is common in the Murray-Darling River Basin within the natural range of these species in Australia during periods of drought, yet it is unknown how well these species tolerate saline conditions. We hypothesized that these turtles would be unable to maintain homeostasis in the 15% water treatment and would suffer osmotic loss of water, increased ionic concentrations and a decrease in body mass. Results revealed that these turtles had elevated plasma concentrations of sodium, chloride, urea and uric acid in the plasma. Plasma ionic concentrations increased proportionally more in E. macquarii than in C. expansa. Individuals of both species reduced feeding in 15% water, indicating that behaviour may provide an additional means for freshwater turtles to limit ion/solute influx when in hyperosmotic environments. This osmoregulatory behaviour may allow for persistence of turtles in regions affected by salinization; however, growth rates and body condition may be affected in the long term. Although we demonstrate that these turtles have mechanisms to survive temporarily in saline waters, it is likely that sustained salinization of waterways will exceed their short- to medium-term capacity to survive increased salt levels, making salinization a potentially key threatening process for these freshwater reptiles.