In the present study, Pseudomonas aeruginosa MK720218 is isolated from 2,4-dinitrotolune (2,4-DNT)-contaminated soil and is investigated for its ability to produce biosurfactant and simultaneously degrade 2,4-DNT present in the soil. Pseudomonas aeruginosa MK720218 reduced both nitro groups on 2,4-DNT and generated 2,4-diaminotoluene (2,4-DAT) with one intermediate metabolite 2-amino-4-nitrotoluene (2A4NT) production. Soil bio slurry experiments demonstrated 2,4-DNT degradation in the presence and absence of rhamnolipid biosurfactants where growth of microorganism, production of nitrite, and kinetics of 2,4-DNT degradation were measured. Enhanced DNT biotransformation was observed over a period of 10 days with rhamnolipids derived from Mahua oil compared with experiments without biosurfactants. It is further shown that Pseudomonas aeruginosa MK720218 cannot utilize DNT as a sole carbon or nitrogen source and requires additional carbon source and nitrogen source for enhanced biodegradation.