The Australian Government has invested $35.4M in the Geological and Bioregional Assessment (GBA) program to determine the risks associated with shale and tight gas development (collectively referred to as unconventional gas from hereon) in 3 gas basins, including the Beetaloo Sub-basin in the Northern Territory of Australia. One of these risks relates to potential adverse effects of unconventional gas flowback/produced waters (FPWs) on freshwater biota. In this study, we performed chronic direct toxicity assessments (DTAs) on a suite of 8 freshwater species (a microalga, aquatic plant, nematode, water flea, midge, snail, shrimp and fish) using FPWs collected from horizontally-drilled and vertically-drilled wells that had been recently hydraulically fractured for exploration in the Beetaloo Sub-basin. We also performed a chronic microalgal test on the initial flowback water from the horizontal well to compare toxicity of FPWs at different phases of production. The chemical characterisation of the FPWs reflected differences in the geogenic components from the different target formations. The FPWs were more toxic than the less saline initial flowback water that contained the highest concentration of hydraulic fracturing fluid additives (guar gum and biocide) as well as volatile hydrocarbons. A significant portion of the chronic toxicity from the FPWs was determined to be due to major ions (Na, Cl-, HCO3-) when toxicity was compared to salinity controls that had matching major ion concentrations to the FPWs. Species sensitivity distributions were used to derive “safe” dilutions that would protect 95% of freshwater biota. The knowledge, data and tools from this work will assist industry, regulators, landowners and the community to make evidence-based decisions on the management of potential impacts of unconventional gas FPWs on the environment in the unlikely event of accidental spills/seepage and the eventual treatment and re-use of FPWs.