The biodegradation of rapeseed oil methyl ester (RME) in pure and in mixtures with diesel fuel was investigated. Higher ratio of diesel fuel in the mixture resulted in higher count of bacteria. Fungal growth was advanced by higher RME contents. The growth of microorganisms gained from soil was strongest in B 20 (20 vol.% biodiesel and 80 vol.% diesel fuel) mixtures followed by B 5 (5 vol.% biodiesel and 95 vol.% diesel fuel) mixtures and pure RME. The formation of free fatty acids (FFA) in the RME sample was measured according to DIN EN 14214. The content of FFA in inoculated RME samples rose from 0.08 mass% to 0.344 mass% at the beginning. The oxidation stability of inoculated samples of B 20, B 5 and pure RME decreased faster than the oxidation stability of blank samples. An optical evaluation showed the formation of turbidity. Partly, the formation of sediment was observed, especially in B 20 and B 5 samples.