Elevated manganese (Mn) is a common contaminant issue for mine water discharges, and previous studies have reported that its toxicity is ameliorated by H+, Ca2+, and Mg2+ ions. In the present study, the toxicity of Mn was assessed in a high risk scenario, that is, the slightly acidic, soft waters of Magela Creek, Kakadu National Park, Northern Territory, Australia. Toxicity estimates were derived for 6 tropical freshwater species (Chlorella sp., Lemna aequinoctialis, Amerianna cumingi, Moinodaphnia macleayi, Hydra viridissima, and Mogurnda mogurnda). Low effect chronic inhibition concentration (IC10) and acute lethal concentration (LC05) values ranged between 140 μg L–1 and 80 000 μg L–1, with 3 of the species tested (M. macleayi, A. cumingi, and H. viridissima) being more sensitive to Mn than all but 1 species in the international literature (Hyalella azteca). A loss of Mn was observed on the final day for 2 of the H. viridissima toxicity tests, which may be a result of the complex speciation of Mn and biological oxidation. International data from toxicity tests conducted in natural water with a similar physicochemistry to Magela Creek water were combined with the present study's data to increase the sample size to produce a more reliable species sensitivity distribution. A 99% protection guideline value of 73 μg L–1 (33−466 μg L–1) was derived; the low value of this guideline value reflects the higher toxicity of Mn in slightly acidic soft waters.