Diesel oil was biodegraded by pure bacterial and yeast strains as well as by their mixtures. The direct impact of natural surfactants (rhamnolipides, saponin) on the biodegradation acceleration and the cell surface hydrophobicity were in scope. Rhamnolipides proved to be the most effective surfactant (150 mg/l). Their addition resulted in reduction of the cell hydrophobicity as compared to cells, which were not exposed to contact with rhamnolipides. However, the rhamnolipides did not show any influence of the cell feature. Mixtures of yeasts with bacterial strains performed extremely well the diesel oil biodegradation degree in each case and were even higher then the separate species.