The effect of the biosurfactant rhamnolipid on phenanthrene biodegradation and cell growth of phenanthrene degraders was investigated. To compare the effect of rhamnolipid addition, two bacterial strains, 3Y and 4-3, which were isolated from a diesel-contaminated site in Korea, were selected. Without the biosurfactant, large amounts of phenanthrene were degraded with both strains at neutral pH, with higher rates of phenanthrene degradation when the cell growth was higher. Upon the addition of 240 mg/L rhamnolipid, the phenanthrene degradation and optical density were reduced, with this inhibitory effect similar for both 3Y and 4-3. To explain this inhibition, the cell growths of both strains were monitored with various concentrations of rhamnolipid, which showed significant toxic effects toward strain 3Y, but was nontoxic toward 4-3. Combining the inhibitory and toxicity results with regard to the biodegradation, different mechanisms can be suggested for each strain. In the biodegradation experiments, the toxicity of rhamnolipid itself mainly was responsible for the inhibitory effect in the case of 3Y, whereas the toxicity of solubilized phenanthrene or the increased toxicity of rhamnolipid in the presence of solubilized phenanthrene could have resulted in the inhibitory effect in the case of 4-3. This study demonstrated that the effectiveness of biosurfactant-enhanced biodegradation can be significantly different depending on the strain, and the toxicity of the biosurfactant should be considered as an important factor.