Abstract In this study, the activity of the biosurfactant produced by the hydrocarbon-degrading bacteria (HDB) Serratia marcescens was investigated at various temperatures (30–100 °C), pH values (pH 4–10) and salt concentrations (1%–9%). The characterization of crude biosurfactant was performed using Fourier transform infrared spectroscopy (FTIR), thin-layer chromatography and tandem mass spectrometry, while purification was conducted by silica gel adsorption chromatography. The biosurfactant displayed good activity at a high temperature of 70 °C, a pH 7 and a salt concentration of 3%. FTIR identified hydroxyl, acetyl, carbonyl and aliphatic chain chemical functional groups. The purification of the crude biosurfactant resulted in three distinct fractions, i.e., glucose, 1-tetradecyl D-glucuronate (novel biosurfactant fraction) and lipids. The crude biosurfactant produced by the HDB S. marcescens is a novel, promising class of biosurfactant for the removal total petroleum hydrocarbon from contaminated soil. Based on these results, the biosurfactant may be an attractive choice for the oil industry for the decontamination of polluted sludge.