Naphthenic acids (NAs) are predominant compounds in oil sands influenced waters. These acids cause numerous acute and chronic effects in fishes. However, the mechanism of toxicity underlying these effects has not been fully elucidated. Due to their carboxylic acid moiety and the reported disruption of cellular bioenergetics by similar structures, we hypothesized that NAs would uncouple mitochondrial respiration with the resultant production of reactive oxygen species (ROS). Naphthenic acids were extracted and purified from 17-year-old oil sands tailings waters yielding an extract of 99% carboxylic acids with 90% fitting the classical O2-NA definition. Mitochondria were isolated from rainbow trout liver and exposed to different concentrations of NAs. Mitochondrial respiration, membrane potential, and ROS emission were measured using the Oroboros fluorespirometry system. Additionally, mitochondrial ROS emission and membrane potential were evaluated with real-time flow cytometry. Results showed NAs uncoupled oxidative phosphorylation, inhibited respiration, and increased ROS emission. The effective concentration (EC50) and inhibition concentration (IC50) values for the end points measured ranged from 21.0 to 157.8 mg/L, concentrations similar to tailings waters. For the same end points, EC10/IC10 values ranged from 11.8 to 66.7 mg/L, approaching concentrations found in the environment. These data unveil mechanisms underlying effects of NAs that may contribute to adverse effects on organisms in the environment.