The worldwide occurrence of antibiotic resistance, has triggered the quest for viable alternatives, to the failing antibiotics in common usage. Biosurfactants have been studied for their potential in this regard. The aim of this study, was to characterise a biosurfactant, produced from Pseudomonas aeruginosa strain S16 (accession number OQ734845), shown to possess effective biocontrol activity, against Staphylococcus aureus SO183 at 0.060 g/L. Pseudomonas aeruginosa strain S16 was isolated from soil in a mixed farm, at Ajibode area, Ibadan, Oyo State, Nigeria, with the use of the pour plate method, identified through biochemical tests, and confirmed through molecular studies. Scanning electron microscopy revealed that the biosurfactant possessed a surface composed of, globular and spindle-shaped projections, while energy dispersive x-ray spectroscopy revealed that its elemental composition was mainly oxygen (3.00%), carbon (36.10%), nitrogen (43.00%), phosphorus (3.00%) and sodium (6.60%); the least abundant was manganese (0.48%). High performance liquid chromatography and mass spectrometry, revealed that the monosaccharides, were mainly rhamnose, glucose, xylose and mannose, while the phenol-sulfuric acid method measured their concentration as 0.0047 g/L. Fourier transform infrared spectroscopy, revealed the presence of carboxyl, alcohol, amine and halo functional groups, amongst others. The biosurfactant produced from Pseudomonas aeruginosa strain S16 was a positively charged, manganese-containing, polymeric biosurfactant made up of carbohydrates, lipid and peptide molecules. It was likely a positively-charged, manganese-containing glycolipopeptide/glycolipoprotein, polysaccharide-protein-fatty acid composite, or lipopolysaccharide-protein composite. The manganese probably conferred to it, the potential for antibacterial and diverse bioactivities. From available records, this is the first report of a manganese-containing biosurfactant.