Abstract The biosurfactant's effect on the biodegradation of 4-chlorophenol (4-CP) in the existence of glucose was researched under the circumstances of using unacclimated culture and various sludge ages. The removal efficiencies of chemical oxygen demand (COD) and 4-CP, the growth of biomass and specific substrate removal rates were examined under various operating conditions. When 150 mg/l concentration of 4-CP was applied, glucose and 4-CP degraded in the same period in the unacclimated bioreactors where biosurfactant was added. Nevertheless, the COD removal in the control reactor noticeably decreased and when compared with reactors which biosurfactant was added, a longer period was needed for the degradation of 4-CP in this reactor. While the complete removal of 4-CP in the control reactor eventuated on the 14th day, in the reactor which 2 × critical micelle concentration (CMC) was added the complete removal of 4-CP eventuated on the end of the 1st day. These results showed that addition of biosurfactant reduced the transient time before the steady-state. COD and 4-CP removal performances were improved by increasing the sludge age. No difference in system performance was observed at high sludge ages in the absence and presence of biosurfactant. However, the performance of the system in the presence of biosurfactant was satisfactory even at low sludge ages. That is, the system should be operated either at high sludge ages (>15 days) in the absence of biosurfactant or at low sludge ages (<15 days) in the presence of surfactants.