The study was carried out to compare the the production capabilities and the biosurfactant activity of the bacteria, Bacillus subtilis and Pseudomonas aeruginosa using engine oil and diesel as the substrates respectively. The test organisms were isolated from engine oil contaminated soil as in the case of the Bacillus subtilis, which was collected from an automobile workshop in Samaru, Zaria and hydrocarboncontaminated water in the case of the Pseudomonas aeruginosa. The medium used for the experiment was a mineral medium supplemented with 2% engine oil and 2% diesel as the sole source of carbon and energy for Bacillus subtilis and Pseudomonas aeruginosa respectively. Production of biosurfactant was assayed by monitoring the increase in cell concentration, biosurfactant concentration, emulsification index and decrease in surface tension. Highest level of cell concentration and biosurfactant concentration (3.3 x 10 8 CFU/ml and 0.0106mg/ml respectively) were obtained at 144 h for the Pseudomonas aeruginosa using diesel as source of carbon and energy while the highest level of cell concentration and biosurfactant concentration (3.2 x 10 8 CFU/ml and 0.0096mg/ml respectively) were obtained at 120hrs for the Bacillus subtilis using engine oil as source of carbon and energy. The research show that Pseudomonas aeruginosa using diesel as the sole source of carbon and energy is better for the production of biosurfactant than Bacillus subtilis using engine oil as the sole source of carbon and energy.