Naphthenic acids are naturally-occurring, aliphatic or alicyclic carboxylic acids found in petroleum. Water used to extract bitumen from the Athabasca oil sands becomes toxic to various organisms due to the presence of naphthenic acids released from the bitumen. Natural biodegradation was expected to be the most cost-effective method for reducing the toxicity of the oil sands process water (OSPW). However, naphthenic acids are poorly biodegraded in the holding ponds located on properties leased by the oil sands companies. In the present study, chemical oxidation using ozone was investigated as an option for mitigation of this toxicity. Ozonation of sediment-free OSPW was conducted using proprietary technology manufactured by Seair Diffusion Systems Inc. Ozonation for 50 min generated a non-toxic effluent (based on the Microtox bioassay) and decreased the naphthenic acids concentration by ∼70%. After 130 min of ozonation, the residual naphthenic acids concentration was 2 mg l−1: <5% of the initial concentration in the filtered OSPW. Total organic carbon did not change with 130 min of ozonation, whereas chemical oxygen demand decreased by ∼50% and 5-d biochemical oxygen demand increased from an initial value of 2 mg l−1 to a final value of 15 mg l−1. GC–MS analysis showed that ozonation resulted in an overall decrease in the proportion of high molecular weight naphthenic acids (n ⩾ 22).