Recently, there has been a push to develop bioavailability-based water quality guidelines for metals that take into consideration the background water chemistry. Dissolved organic matter (DOM) is a key parameter that influences the toxicity and bioavailability of metals to aquatic organisms and is being considered for inclusion in the development of bioavailability models. Most research to date have utilised purchased DOM in the form of humic acid derived from leonardite or DOM isolated from freshwaters in the Northern Hemisphere, which may not be representative of DOM in Australian freshwaters. This study compares the influence of DOM isolated from different Australian waterways on the toxicity and bioavailability of three metals of concern: Cu, Ni and Zn to a freshwater alga Chlorella sp. and compares results to key DOM characteristics. Toxicity and bioavailability of Cu was shown to decrease with increases in DOM in the presence of all DOMs tested, with more aromatic DOM of higher molecular weight, higher in humic-like components generally more protective. In contrast DOM did not have a great influence on Ni toxicity and bioavailability, and depending on DOM type, significantly increased toxicity of Zn. This suggests that the protective ability of DOM is DOM- and metal-specific and this needs to be considered when developing appropriate risk assessment tools.