Staphylococcus aureus and coagulase-negative staphylococci comprises approximately 65% of infections associated with medical devices and are well known for their biofilm formatting ability. Currently, there is no efficient method for early biofilm detection. Therefore, we aimed to construct a comprehensive reference map followed by identifying marker proteins in the different growth phase of hydrated biofilm, and then perform pathway analysis, subcellular localisation and protein-protein interaction network mapping. S. aureus reference strain (ATCC 25923) was grown in tryptic soy broth to produce a 24-hour planktonic, 3-day wet biofilm (3dwb), and 12-day wet biofilm (12dwb). The Centres for Disease Control biofilm reactor was used to grow biofilms. Tandem Mass Tag (TMT)-based mass spectrometry was performed, and protein identification and relative quantitation of protein levels were performed using Proteome Discoverer (version 1.3). Statistical analysis was done using the TMTPrePro R package. We identified 1636 total biofilm extractomes. Among the significantly (˃2-fold) up-regulated proteins in 3dwb, hyaluronidase encoded by hysA an extracellular enzyme involved in dispersing established biofilms by degradation of hyaluronic acid. Proteins significantly up-regulated in 12dwb are mostly involved in energy metabolism and cell wall formation. Among the significantly down-regulated proteins, chitinase encoded by SA0914 an extracellular enzyme involved in quorum sensing and responsible for preventing initial attachment of biofilm formation. Interestingly, chitinase can hydrolyse N-acetyl-D-glucosamine which is the structural component of hyaluronic acid. Therefore, hysA in combination with chitinase may play a potential role in the eradication and/or prevention of biofilm formation. In addition, protein-protein interaction network showed significantly more interactions for 3dwb and 12dwb. Current study showed a significant range of quantitative proteomic shifts and changes in metabolic process in biofilm. HysA and chitinase may be potential targets in the biofilm research in vitro and in vivo. The proteins identified might be helpful in designing advanced targeted candidates for vaccines, anti-biofilm agents, diagnostic biomarkers for S. aureus biofilm-related infections associated with implantable medical devices.