Several biosurfactant-producing bacterial strains were isolated from petroleum-contaminated soil. The isolate ADMT1, identified as a new strain of Pseudomonas aeruginosa, was selected for further studies on the basis of oil displacement test and emulsification index (E24). The optimal parameters for production, determined by employing Box-Behnken design, were temperature 36.5 C and pH 7. The environmental isolate ADMT1 produced significant amount of biosurfactant (1.7 g L −1 in 72 h) in minimal salt medium (MSM) using dextrose as the sole carbon source. The E24 value and critical micelle concentration (CMC) of the biosurfactant was 100% and 150 mg L −1 , respectively. At CMC, the surface tension of water was reduced to 28.4 mN m −1 . The biosurfactant exhibited hemolytic activity and antibacterial activity against 8 reference strains of pathogenic bacteria, including 2 methicillin-resistant Staphylococcus aureus strains (MRSA ATCC 562 and MRSA ATCC 43300), with minimum inhibitory concentration (MIC) of 0.4 and 0.2 mg mL −1 , respectively. The structure of biosurfactant was characterized by FTIR, 1 H, and 13 C NMR spectroscopy. 7 di-rhamnolipid (RL) congeners were identified in the biosurfactant by ultraperformance liquid chromatographymass spectrometry analysis. The major congeners, which constituted 67% of the RL mixture, included Rha-Rha-C 10