Reactive oxygen species (ROS) production and resultant oxidative stress (OS) has been implicated as a pathway of toxicity in animal species exposed to pollutants. The gills of aquatic animals and the liver and kidneys of mammalian species are specific cellular sites of toxicity. Oxidative effects of acid mine drainage effluent (following passive and active treatment) impacting a natural stream were assessed using selected cell lines. Levels of pollutants such as heavy metals in acid mine drainage (AMD) effluent can be quantified following treatment, but it is unknown whether this is associated with equivalent reduction in toxicity. ROS production by AMD untreated (U) and after treatment (T) was quantified in a fish gill cell line (RTgill-W1) and in two mammalian cell lines (C3A human liver and Vero monkey kidney). ROS production was determined using the oxidant sensitive fluorogenic probe, 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) following exposure to U and T, AMD water. Treatment of AMD water caused reduction in levels of Al, Zn, Fe, Si and Mn while levels of Cr, Cu, Ar and Hg remained unchanged. A dose-dependent increase in ROS production was observed for U and T. ROS formation decreased from 14% to 4.5%, 16.4% to 7.2% and 25.3% to 17.7% in the RTgill-W1, C3A, and Vero cell lines exposed to 100% AMD water, U and T. The presence of Mn and/or other ions in treated water and subsequent ROS formation indicates that water could still be toxic to cells and requires further processing. The DCFH-DA assay in several cell lines can be used to rapidly bio-monitor quality of AMD water related to formation of ROS and subsequent cellular effects. However, cut-off levels for cellular toxicity must be established to ensure safety of this water for aquatic animals and for animal and human consumption.