Naphthenic acids (NAs) have a variety of commercial uses including as emulsifiers and wood preservatives. They have been identified as being the main component responsible for the acute toxicity in produced waters in the oil sands operations in northeastern Alberta, Canada. NAs comprise a complex mixture of alkyl-substituted acyclic and cycloaliphatic carboxylic acids, with the general chemical formula CnH2n+ZO2, where n indicates the carbon number and Z specifies hydrogen deficiency from ring formation. In this study, commercial preparations of NAs were shown to be degraded in aerobic cultures from oil sands process-affected waters. High-performance liquid chromatography and gas chromatography−mass spectrometry (GC−MS) were used to monitor the concentrations and composition of the NA mixtures during biodegradation. Within 10 days of incubation, the NAs concentrations dropped from about 100 to <10 mg/L. This was accompanied by the release of about 60% of carbon from the NAs as CO2 and the reduction of toxicity of the culture supernatant, as measured by the Microtox assay. GC−MS results demonstrated that biodegradation changes the composition of the complex mixture of these NAs and that the lower molecular weight acids (with n = 5−13) were degraded more readily than the high molecular weight acids.