In the present study a vancomycin resistant Staphylococcus aureus (S. aureus) (VRSA) (Labeled as CP2) was isolated from the blood of a post-operative cardiac patient is described. It harbors a plasmid which carry vanA gene and exhibited low-level vancomycin resistance (MIC 16μg/mL), was sensitive to teicoplanin. It has been observed that sub-lethal dose of vancomycin induced biofilm formation by CP2 on nylon and silicon indwelling. The results divulge new insights into associations between vancomycin induced biofilms and extra-cellular fatty acids. Gas chromatography coupled with mass spectrometry (GC-MS) revealed that biofilm matrix of CP2 contains a variety of saturated and un-saturated fatty acids, especially, diverse species of octadecanoic (C18:0) and octadecenoic acids (C18:1). A large difference in fatty acids composition was noticed in biofilms, isolated from hydrophobic and hydrophilic surfaces. CP2 produced thicker layer of biofilms on hydrophobic silicon and nylon surfaces which contains variety of saturated, un-saturated and cyclic fatty acids. Contrary to this on hydrophilic glass surfaces it produced thinner layer of biofilm which contains only straight chain saturated fatty acids. These fatty acid components seem to play a crucial role in cell-cell communication and in the establishment of biofilms, consequently, advantageous for pathogens to survive in hospital environment under enormous antibiotics pressure.