In this study, adsorption of dirhamnolipid biosurfactant on a Gram-negative Pseudomonas aeruginosa, two Gram-positive Bacillus subtilis, and a yeast, Candida lipolytica, was investigated, and the causality between the adsorption and change of cell surface hydrophobicity was discussed. The adsorption was not only specific to the microorganisms but also depended on the physiological status of the cells. Components of the biosurfactant with different rhamnosyl number or aliphatic chain length also exhibited slight difference in adsorption manner. The adsorption indeed caused the cell surface hydrophobicity to change regularly; however, the changes depended on both the concentrations of rhamnolipid solutions applied and the adsorbent physiological conditions. Orientation of rhamnolipid monomers on cell surface and micelle deposition are supposed to be the basic means of adsorption to change cell hydrophobicity at low and high rhamnolipid concentrations, respectively. This study proposed the possibility to modify cell surface hydrophobicity with biosurfactant of low concentrations, which may be of importance in in situ soil remediation.