The inherent stability and solvent processability restrict the functional expression of graphene for biomedical applications. To resolve this problem graphene oxide (GrO) has emerged as a biomaterial for biomedical applications of graphene. The participation of O_K edges has been confirmed with synchrotron radiation near edge X-ray absorption fine structure (SR-NEXAFS). The as-prepared graphene oxide has been confirmed with ¹³C cross polymerization magic angle spinning (CP-MAS) solid state NMR, HRXPS, HRTEM, SAED, SEM, AFM, Raman, XRD, FTIR, UV, TGA, DTA, BET and Porosimeter, etc. The biomedical significance of graphene oxide has been supported by an extensive structural investigation. The underlying mechanism of biological activity of graphene oxide has been inferred through the synergistic impact of contributory factors (SICF). The cytotoxicity of graphene oxide has been evaluated with an SRB assay with living mammalian cell lines, human breast cancer cell line MCF-7 and monkey normal kidney cell line Vero. The estimated cell viabilities are greater than 80% over a 10–80 μg mL⁻¹ concentration. The high cytocompatibility of graphene oxide has confirmed its low toxicity and hence its excellent biocompatibility. Moreover, the morphological effect on the Vero cell line has been endorsed by in vitro behaviour. The sensitivity of graphene oxide has been evaluated with minimum inhibitory concentration (MIC) against antifungal strain C. albicans (ATCC 10231) at 128 μg mL⁻¹. However, for antibacterial strains S. aureus (ATCC 25923) and E. coli (ATCC 25922) the MIC of graphene oxide is 256 μg mL⁻¹. The genetic variability greatly increases during the sub-culturing of strains; therefore the resistant behaviour of graphene oxide has been observed against NCIM strains. Additionally, the antioxidant activity of graphene oxide is 50.57–45.89%, at over 40–100 μg mL⁻¹ concentration. Hence, the structural investigations of graphene oxide have complemented the findings for biological activity and emerged as an advanced functional biomaterial for biomedical applications of graphene.