This study was conducted to improve the voltage production, desalination, and COD removal efficiencies of a five-chamber Microbial Desalination Cell (MDC). To do this, rhamnolipid was added to anolytes only and catholytes stirred to determine the effects of these factors on the MDC activity. This was followed by a factorial study to investigate the effects of the interactions of rhamnolipid and stirring on the voltage production, desalination, and COD removal efficiencies of the MDC. Increasing the concentration of rhamnolipid to 240 mg/L improved the peak voltage produced from 164.50 ± 0.11 to 623.70 ± 1.32 mV. Also, the desalination efficiency increased from 20.16 ± 1.97 % when no rhamnolipid was added to 24.89 ± 0.50 % at a rhamnolipid concentration of 240 mg/L, and COD removal efficiency increased from 48.74 ± 8.06 % to 64.17 ± 5.00 % at a rhamnolipid concentration of 400 mg/L. In the stirring experiments, increasing the number of stirring events increased peak voltage from 164.50 ± 0.11 to 567.27 ± 18.06 mV. Similarly, desalination and COD removal efficiencies increased from 20.16 ± 1.97 % and 48.74 ± 8.06 % to 24.26 ± 0.97 % and 50.23 ± 1.60 %, respectively, when the number of stirring events was more than twice a day. In the factorial study, voltage production, desalination, and COD removal efficiencies were 647.07 mV, 25.50 %, and 68.15 %, respectively. However, the effect of the interaction between rhamnolipid and stirring was found to be insignificant (p>0.05). Thus, the addition of only rhamnolipid or the stirring of catholytes only can improve the performance of the five-chamber MDC.