Biosurfactants, produced by microorganisms, are surface-active compounds with abundant applications in miscellaneous industries like food, cosmetics, bioremediation, agriculture, and so on. The primary objective of this study was to investigate the larvicidal activity of biosurfactants produced by Staphylococcus epidermidis (S. epidermidis) isolated from petrochemical wastes. As the secondary objective, this study was to perform a phylogenetic analysis of this bacterium. To this end, the type of the extracted biosurfactant was characterized using the thin-layer chromatography (TLC) and the gas chromatography/mass spectrometry (GC-MS). The isolated bacterium was then identified through molecular tests based on 16S ribosomal ribonucleic acid (16S rRNA) and serum response factor (srf ) genes. Likewise, the phylogenetic tree of the bacterium was deduced via the Molecular Evolutionary Genetics Analysis (MEGA-X) software. Besides, the larvicidal activity of the biosurfactant on the larvae of flour beetles, Tribolium castaneum (T. castaneum), was examined. The mean mortality rate and the lethal dose (LD50) were further calculated using the Statistical Analysis System (SAS) and the Probit Analysis-MSChart software. Totally, nine diverse types of colonies were isolated and three of them were found with potential activity to produce biosurfactants. The best bacterium was accordingly selected for subsequent experiments recruiting the oil displacement process and the emulsification index (E24). The extracted biosurfactant was then characterized as a lipopeptide. Moreover, biochemical tests showed that the bacterium was Gram-positive cocci of gamma species. Hemolysis and molecular tests also confirmed that the bacterium was S. epidermidis. As well, the presence of the SRF gene in the isolation was established via the polymerase chain reaction (PCR). The mean mortality rate revealed that the mentioned bacterial biosurfactant at the concentration of 10000 μg/g and on the ninth day of the treatment had the highest larval mortality rate (63.33%). In addition, the LD50 equal to 25001.7 μg/g was calculated for S. epidermidis on the ninth day. The results demonstrated that the biosurfactant produced by this bacterium was endowed with an acceptable ability to control T. castaneum and it could be used in further research on the larvae of other insects.