The main objective of the present work is the screening of biosurfactant producing hydrocarbonoclast bacteria from oil production waters of the Sidi LITAYEM field. The screening allowed the isolation of two bacterial strains B17 and S7 with strong capacities to produce biosurfactants on two different carbon sources (olive oil and motor oil). These results were confirmed by measurements of percent emulsifying activity and oil displacement diameters. Indeed, E24 in the range of 44% and 33% and diameters equal to 5.4 and 5 cm were obtained by the biotensioactives produced by the B17 and S7 strains respectively. The study of the stability of the biosurfactants towards temperature, pH and salinity, showed that the biosurfactant B17 is more stable at elevated temperatures up to 100 °C and was found to be resistant to hypersalinity (100 g/L) and acidity of the medium (pH = 4). The study of biological activities revealed that the biosurfactants B17 and S7 show relatively good antibacterial activity against the different pathogenic and phytopathogenic strains tested with MICs of 4.993 m