The bacterial strains from the genera: Bacillus, Pseudomonas, Aeromonas, Achromobacter, and Flavimonas isolated from soil contaminated with crude oil were the subject of studies. The effect of the addition of rhamnolipids on cell surface properties and the removal efficiency of diesel oil were investigated. Rhamnolipids caused the modification of cell surface properties of tested strains, which depended on the amount of external additions of biosurfactant. Additionally, the decrease of Zeta potential was observed after the introduction of rhamnolipids to the diesel oil system. Particle size distribution provides information about system homogeneity and the tendency of particles toward agglomeration. Cell surface hydrophobicity during hydrocarbon biodegradation is a dynamic parameter. There were no different effects, after the addition of rhamnolipids, on the Gram positive and Gram negative bacterial strains. Moreover, the addition of rhamnolipids does not always increase the efficiency of diesel oil biodegradation. The most effective bacterial strain in diesel oil removal was the genus Pseudomonas.