Biodiesel, a mixture of fatty acid methyl esters (FAMEs), is increasingly recognized as a renewable fuel. While some environmental impacts of biodiesel usage have been investigated, accumulation of organic compounds in motor oil, which can subsequently leak onto roads, has not been studied. Because studies have shown that toxic polycyclic aromatic hydrocarbons (PAHs) accumulate in the motor oil of engines fueled with fossil diesel or gasoline, the objective of this study was to determine if this also occurs for engines fueled with biodiesel. Here, we sampled and analyzed motor oil of a biodiesel-powered 2005 Volkswagen Passat Wagon over 3240 km of personal-use driving. Using gas chromatography with flame ionization detection (GC−FID), we found a total of 0.5% FAMEs in the motor oil after 3240 km. We also used gas chromatography−mass spectrometry and comprehensive two-dimensional gas chromatography and did not detect PAHs or other organic compounds not present in the initial motor oil. Using natural radiocarbon analysis, a powerful technique capable of detecting biodiesel-derived carbon that would be otherwise undetectable by gas chromatography, we found a total of 0.68% biodiesel-derived carbon after 3240 km. This is similar to the amount of FAMEs found in these samples with GC−FID, indicating that the primary source of biodiesel-derived carbon in the motor oil is FAMEs (and not PAHs or other carbonaceous species). This result suggests that used motor oil of biodiesel vehicles can be less toxic based on PAH content than vehicles fueled with fossil diesel or gasoline.