The potential of a greener and cheaper substrate (waste cooking oil) in place of diesel for rhamnolipid production in laboratory scale was addressed in this investigation. In shake flasks study, rhamnolipid production was comparable when either diesel or waste cooking oil was used as the main carbon source. The yield was 2.15 g/L and 2.49 g/L for waste cooking oil and diesel, respectively. Rhamnolipid production with waste cooking oil as a carbon source was further studied in a 3.6 L bioreactor at two different agitation speed, 400 and 500 rpm. The agitation speeds were selected based on the similar volumetric mass transfer coefficient (k_La) of 10.8/h, as determined earlier in a non respiring system. 2.23 g/L of rhamnolipid was produced at 400 rpm compared to 1.09 g/L in broth and 1.80 g/L in foam obtained in the same bioreactor agitated at 500 rpm. Thus, waste cooking oil could be utilized as an alternative carbon source for a low cost rhamnolipid production.