Development of highly improved or reinforced Graphene oxide nanosheets were synthesized by converting it into a whole green GO nanofiber material and by infusing the aqueous seed extracts of Sesamum indicum (SI), along with the fiber generation process of GO. The synthesized GO nanofibers (NF) and GO/SI NF were characterized for structural, morphological, elemental and functional group analysis. The antimicrobial and biocompatible nature of GO/SI NF was evaluated in comparison with GO NF. The results showed the non-toxic nature of both the nanofibers (GO NF and GO/SI NF) on gram-negative bacteria (Klebsiella oxytoca) and gram-positive bacteria (Staphylococcus aureus). Both the nanofibers were hemo-/biocompatible toward human red blood cells and human epithelial cell lines (TH 1); however, the hemo-/biocompatibility was further increased after the addition of SI into GO NF, and therefore, GO/SI NF can be a potent green nanofiber material for the biomedical applications.