In E.faecalis, fsr two-component signal transduction system is a well-defined quorum sensing system which regulates biofilm formation by gelatinase production [1]. Previous studies on enterococcal biofilm have suggested several other biofilm associated factors including enterococcal surface protein (esp) [2], aggregation substance [3], pili [4], autolysin [5] and D-alanylation of Lipoteichoic acid (dltABCD) [6]. Several studies on E. faecalis comparing biofilm formers and non-biofilm formers have identified protein translation machinery, aromatic amino acid biosynthesis and sugar and sulfate permease transporter systems to have a momentous role in biofilm formation [7, 8]. E. faecalis SK460 used in the present study is isolated from a chronic diabetic ulcer patient [9] and is devoid of several well-defined biofilm associated factors including fsr quorum signaling, gelatinase production and enterococcal surface protein. Lack of these biofilm determinants does not affect the biofilm forming potential of SK460. This led us to focus on the role of differential protein expression pattern in biofilm phenotype of this strong biofilm former. The present study utilized label-free quantitative approach to decipher the protein expression pattern of E. faecalis SK460 at planktonic and biofilm stages to elucidate the unexplored links in understanding the enterococcal biofilms. This helps to deliver the comprehensive knowledge regarding the metabolic pathways and cellular processes involved in Enterococcal biofilm to come up with potential biofilm inhibiting targets.