Estuaries are unstable ecosystems and can be changed by the environmental and anthropogenic impact. The Murray Estuary and Coorong were degraded by drought and low freshwater input in the last decade and therefore transformed into the largest hyper-saline lagoon in Australia. This study evaluates the physiological stress of two estuarine fish species (small-mouthed hardyhead Atherinosoma microstoma and Tamar goby Afurcagobius tamarensis) to the induced salinity change in captivity. The test fishes were collected from the Coorong and transported to the laboratory in the water from the Coorong. Each fish species was exposed to different levels of salinity, and a number of enzymes were assessed to measure the stress response of fish to salinity change. The activity of reactive oxygen species was significantly increased with the salinity change in both fish species compared with the fish in the control. Significant salinity effect on superoxide dismutase activity was observed on Tamar goby but not on small-mouthed hardyhead. Conversely, the impact of salinity on catalase activity was detected on small-mouthed hardyhead but not on Tamar goby. The study reveals that the induction of physical stress by salinity changes occurred in both Tamar goby and small-mouthed hardyhead despite the varying response of antioxidant enzymes between fish species. The study provides an insight into the understanding of physiological adaptation in estuarine fish to salinity change. The results could improve our knowledge on stress response and resilience of estuarine fish to hypo- and hyper-salinity stress.