Environmental pollution from hydrocarbons and their derivatives is predominantly of anthropogenic origin, occurring mainly during mining, transportation, and storage. This often in an annual release of substantial amounts of pollutants into the environment. The effective restoration of petroleum-contaminated environments requires a series of interventions to remediate soil to its natural state. This study aimed to isolate and characterize biosurfactant-producing bacterial strains with potential bioremediation capabilities to assess the potential application for remediating soils polluted with spent engine oil in the Remo district, Ogun State. Bacterial isolates were recovered from soil samples collected from spent engine oil contaminated sites, using standard cultural techniques and characterized by biochemical and molecular techniques (16S rRNA amplification and sequencing). The isolates were further screened for biosurfactant activity, and the biosurfactant extract was characterized using gas chromatography-mass spectrometry (GC-MS) and Fourier-transform infrared (FTIR) spectroscopy. All 36 isolates recovered from the samples were positive for blood hemolysis, oil spread, and emulsification index assays. Pseudomonas sp. and Klebsiella sp. were identified as the most promising biosurfactant-producing isolates by 16S rRNA gene sequencing by the screening results. The GC-MS and FTIR spectroscopy of the biosurfactant extracts produced by Pseudomonas sp. revealed distinct peak values with various functional groups. FTIR analysis identified various functional groups, including aliphatic ether, ester, sulfone, alkene, allene, isothiocyanate, and alcohol, as the major compounds present in the biosurfactant. 20 bioactive compounds with different peak values were identified by GC-MS analysis. The rhlA gene associated with rhamnolipid production was detected in the two characterized P. aeruginosa strains. Subsequent laboratory-scale bioremediation of spent engine oil-contaminated soil demonstrated a significant (53%) removal of oil. The results highlight the potential applicability of eco-friendly biosurfactants for contaminated soil cleanup and suggest the need for further exploration, optimization, and elucidation of the genetic basis of biosurfactant production for improved efficacy.