Dissolved oxygen levels low enough to impact upon aquatic fauna are common, especially in warm tropical wetlands. Dissolved oxygen is one of the most commonly measured water quality parameters in the field, but the lack of knowledge of faunal tolerances to low dissolved oxygen (termed hypoxia) restricts interpretation of the observed concentration values. We tested the laboratory hypoxia tolerance of 15 fish and one crustacean species from northern Australian freshwaters. Our rapid testing protocols allow a large number of tests to be completed in a relatively short time frame and allows for video recording of various behavioural and physiological responses to progressive hypoxia. The tests included gill ventilation rate tests and relative oxygen consumption rate, demonstrating lethal and sub-lethal effects on fishes across a range of size classes. Many fish species have surprisingly low lethal thresholds, often below 20% or even 10% saturation. The most sensitive species tested were banded grunter, fly-specked hardyheads, eastern rainbowfish and Pacific blue-eyes. Fish size and water temperature both had significant effects on tolerance levels. Data from these tests has been used to recommend revised water quality guidelines for dissolved oxygen with default chronic and acute effect trigger values of 75% and 30% saturation respectively.