Biosurfactants are one among the best alternative for synthetic surfactants that are exploited by many researchers. Several agro wastes help to reduce the cost biosurfactants by being renewable and economical. The present research focuses on the biosurfactant production from Pseudomonas mosselii utilizing Parthenium hysterophorus as a relatively cheap substrate. P. hysterophorus being a hazardous weed, its eradication is quite tedious. So, the utilization of the weed for useful purposes serves as a choice to overcome the problems posed by the weed. In the study, this weed has been successfully utilized as a substrate and the optimized fermentative production of biosurfactant was done. From one-factor-at a-time analysis it was known that the substrate level of 3% incubation time of 96 h, pH 6.0, temperature 35 °C, glucose and yeast extract was found to be the best C and N sources for a high yield. The extracted biosurfactant was partially purified and characterized using FTIR. The biosurfactant produced from the weed could help to render the milestone for distinct biomedical and other applications.