The aim of this present study is to investigate the antimicrobial activity of rhamnolipid biosurfactant produced by Pseudomonas fluorescens MFS03 isolated from mangrove forest soil using groundnut oil cake as substrate. The biosurfactant was extracted with an equal amount of ethyl acetate and the concentrated extract was subjected to FT-IR analysis. The important adsorption bands at 3466.24, 2926.45, 1743.47, 1407.30 and 1162.26 cm-1indicate the chemical structure of rhamnolipid. The rhamnolipid biosurfactant was investigated for the potential antimicrobial activity by using disc-diffusion method against Gram positive bacteria (Bacillus subtilis, Listeria monocytogenes, Staphylococcus aureus, Methicillin resistance S. aureus) Gram negative bacteria (Escherichia coli, Salmonella typhimurium) and a yeast (Candida albicans). The biosurfactant showed distinct antibacterial activity towards tested bacteria and shows an antifungal activity against yeast. The biosurfactant with different concentration was performed for the evaluation of antimicrobial activity. Maximum antimicrobial activity of the biosurfactant (50μl) was observed in S. aureus (23 mm) and it was found that the biosurfactant activity was dependent on the concentration. So it could be used as a therapeutic agent in pharmaceutical application.