An active biosurfactant-producer from an agronomic environment majorly from the rhizosphere of a wheat plant in western Nigeria was utilized for the production of rhamnolipid. The isolated bacteria were selected using different methods such as oil displacement test, emulsification activity, and surface tension respectively. Based on partial sequenced 16S rDNA analysis of isolate, C1501 was identified as Pseudomonas aeruginosa with an accession number KF976394 having 100% similarity to P. aeruginosa LMG 1242T. The results showed that Pseudomonas aeruginosa has the ability to grow and reduce surface tension under a wide range of pH and carbon source. It was observed that strain C1501 reduced the surface tension of glycerol than glucose at the various concentration tested. The surface tension of 61.2 dynes/cm was also reduced to 30 dynes/cm at 12 h after the inoculation. The biosurfactant obtained from strain C1501 was purified and characterized using TLC, Liquid
Chromatography/Mass Spectrometry (LC–MS) and nuclear magnetic resonance spectroscopy (NMR). The gravimetric analysis showed that strain C1501 efficiently biodegrade the tested crude oil with the following degradation percentage of 33%, 71.3% and 96% on 5th, 10th and 20th day respectively. The biosurfactant did not exhibit an inhibitory effect on the seed of economic crops tested, but demonstrated some broad antimicrobial activities against gram positive, gram negative and rot-inducing fungus when compared to the synthetic surfactant from Tween 20. New isomers and congeners rhamnolipids were detected from the Liquid Chromatography/Mass Spectrometry (LCMS). The predicted structure of the rhamnolipid was found to be Lrhamnosyl- L-rhamnosyl-3-b-hydroxydodecenoate. The study suggests application of the strain C1501 biosurfactant as an appropriate candidate for many applications such as biomedical, food industries, agriculture, and bioremediation