Biosurfactants have been found capable of replacing synthetic surfactants which include ongoing bioprospecting of biosurfactant-producing bacteria as well as process optimization for maximum biosurfactant production. In this study, five morphologically distinct actinomycete strains isolated from hydrocarbon-polluted soil collected from an oil spill surface in Southeastern Algeria were tested for their ability to produce biosurfactants using preliminary biosurfactant screening assays. The 7SDS strain was selected as the most promising biosurfactant producer due to its greatest oil displacement diameter (7.83±0.15 cm), emulsification index (59.66±0.44%), and enhanced surface tension reduction (30.04±0.51 mN/m); it was identified as Streptomyces thinghirensis 7SDS using 16S rDNA sequence analysis. The 7SDS strain’s biosurfactant production was optimized using the Face-centered central composite design (CCD) based on response surface methodology (RSM). To this end, five independent factors, i.e., residual frying oil, used engine oil, whey, CS filtrate, and incubation time, were assessed. The RSM’s model predicted a surface tension of 27.48 mN/m using 2.44% (v/v) residual frying oil, 0.35% (v/v) used motor oil, 0.83% (v/v) whey, 0.39% (v/v) CS filtrate, and an incubation time of 219.3 h. The optimized medium produced 8.79 g/L of biosurfactant. The produced biosurfactant allows one to reduce the surface tension of distilled water from 70.86 mN/m to 27.96 mN/m at a critical micelle concentration of 350 mg/L, even over a wide range of pH (2.0–12.0), temperature (4–120 °C), and salinity (2–12%, W/V). Biochemical (Biuret, phenol-sulfuric acid, and phosphate tests) and compositional (FTIR and GC-MS) characterizations confirmed the phospholipid nature of the produced biosurfactant. Interestingly, the produced BS demonstrated significant antimicrobial activity as well as intriguing activity in removing hydrocarbons from polluted soil. Because of their appealing biological properties, strain 7SDS and its biosurfactant are attractive targets for a variety of applications such as biomedicine and environmental ones.