Sixteen biosurfactant-producing bacteria were isolated from multiple petroleum-contaminated soils in Kaluybia Governorate, Egypt. Six screening tests (emulsification index (EI24%), oil spreading assay, emulsification activity, cetyltrimethylammonium bromide (CTAB), hemolytic and lipolytic activities) were conducted to select the high producer isolate. Out of 16 isolates, only one was chosen for molecular identification as the most efficient biosurfactant-producer and was found to be Pseudomonas putida ON763757. The type of biosurfactant was determine via three chromatography analytical methods, High-performance liquid chromatography (HPLC), Fourier-transform infrared spectroscopy (FT-IR) and Gas chromatography–mass spectrometry analysis (GC-MS). HPLC analysis showed that chlorogenic acid (1409.20 µgg-1) was the main component, followed by ellagic acid (530.12 µgg-1). FTIR and GC-MS analyses indicated that the biosurfactant of Ps. putida was rhamnolipid, which is an exciting product for environmental and industrial applications.