Hypoxia represents a growing threat to biodiversity in freshwater ecosystems. Here, aquatic surface respiration (ASR) and oxygen thresholds required for survival in freshwater and simulated blackwater are evaluated for four lowland river fishes native to the Murray-Darling Basin (MDB), Australia. Juvenile stages of predatory species including golden perch Macquaria ambigua, silver perch Bidyanus bidyanus, Murray cod Maccullochella peelii, and eel-tailed catfish Tandanus tandanus were exposed to experimental conditions of nitrogen-induced hypoxia in freshwater and hypoxic blackwater simulations using dried river red gum Eucalyptus camaldulensis leaf litter. Australia's largest freshwater fish, M. peelii, was the most sensitive to hypoxia but given that we evaluated tolerances of juveniles (0.99±0.04 g; mean mass ±SE), the low tolerance of this species could not be attributed to its large maximum attainable body mass (>100,000 g). Concentrations of dissolved oxygen causing 50% mortality (LC50) in freshwater ranged from 0.25±0.06 mg l−1 in T. tandanus to 1.58±0.01 mg l−1 in M. peelii over 48 h at 25–26°C. Logistic models predicted that first mortalities may start at oxygen concentrations ranging from 2.4 mg l−1 to 3.1 mg l−1 in T. tandanus and M. peelii respectively within blackwater simulations. Aquatic surface respiration preceded mortality and this behaviour is documented here for the first time in juveniles of all four species. Despite the natural occurrence of hypoxia and blackwater events in lowland rivers of the MDB, juvenile stages of these large-bodied predators are vulnerable to mortality induced by low oxygen concentration and water chemistry changes associated with the decomposition of organic material. Given the extent of natural flow regime alteration and climate change predictions of rising temperatures and more severe drought and flooding, acute episodes of hypoxia may represent an underappreciated risk to riverine fish communities.