In this study, reduced graphene oxide nanosheets (rGO) were synthesized using a chemical bottom-up technique of modified Hummer’s method. The as-synthesized graphene sheets were characterized optically by Fourier Transform Infra Red spectroscopy (FTIR) and Raman spectroscopy. The morphological characterizations were performed using Field-Emission Scanning Electron Microscopy (FE-SEM), High-Resolution Transmission Electron Microscopy (HR-TEM), Atomic Force Microscopy (AFM) and Confocal Microscopy, respectively. It could be evident from the images that wrinkles and surface corrugation are clearly visible from the morphological analysis. The biomedical applications of rGO nanosheets have been studied in the cancer cell lines of A375 and A549 cells at the in-vitro conditions. MTT assay and live dead assay results clearly indicated that rGO exhibited robust anticancerous activities against the chosen cell lines. Although, it could not elicit any antimicrobial activity at the selected doses. Understanding the primary mechanism and the underlying phenomenon involving the interaction of the rGO nanosheets with the cell systems reveals the potentialities of the nanosheet in designing future-based anticancerous therapeutics at the nanoscale level.