STRING analysis revealed that upregulated genes including stress response factors, major glycolytic enzymes, arginine metabolism and rhamnose biosynthesis were either directly or indirectly poses a close molecular interaction (p-value < 1.0e-16), thereby regulating each other in stressful environment prevailing during biofilm development. Among the upregulated cellular processes, rfbB (Rhamnose biosynthesis), arcA (Arginine deiminase), luxS (Quorum sensing), cAD1 (Pheromone cAD1 lipoprotein) and fbn (Fibrinogen/Fibronectin binding protein associated with adhesion) were selected for gene expression analysis and were found to be enhanced by 2.29, 4.03, 2.35, 1.5 and 13.87 fold respectively in biofilm stages. Of these, luxS mediated quorum sensing system is attributed to play a major role in E. faecalis SK460 biofilm which is devoid of fsr two-component signal transduction system. Accordance of the proteome data with RT-PCR results confirms the reliability of the analysis, serving as a validation for the identified determinants of Enterococcal biofilm development.