Naphthenic acids are components of most petroleums, including those found in the Athabasca Oil Sands of northeastern Alberta. Some naphthenic acids that are solubilized during bitumen extraction from oil sands are acutely toxic to a variety of organisms. Four-month enrichment cultures obtained from the rhizospheres of five plant species native to Alberta, and established with the addition of bitumen (0.5%) as the sole carbon source, revealed a high potential for aerobic degradation of a Merichem commercial preparation of naphthenic acids. Changes in the concentration and composition of the naphthenic acids mixtures during incubation were followed using high-performance liquid chromatography and gas chromatography–electron impact mass spectrometry. Concentrations did not significantly change in the sterile control, but they decreased by up to 90% after 10 days of incubation in the viable cultures. Lower molecular mass naphthenic acids were preferentially degraded, while the proportion of high molecular mass acids increased during incubation. By day 17, the most abundant ions were derived from cellular membranes, corresponding to an increase in microbial numbers in the cultures as naphthenic acids were metabolized. This study is the first to demonstrate the biodegradation potential of microorganisms from rhizosphere soils to biodegrade naphthenic acids.