The rapid growth of graphite market is highly coupled with the increasing demand for Li-ion grade graphite, the production of which results in significant losses of the graphitic material in the form of graphite fines. Herein, for the first time, we report an effective strategy to utilize industrial waste graphite fines through the development of graphene oxide-based nanohybrids as non-toxic and efficient antibacterial agents. To achieve this, graphene oxide (GO) was initially synthesized using industrial waste graphite fines as a graphitic precursor. Subsequently, hyperbranched polyethyleneimine (PEI), or either of its guanidinylated (GPEI) and N-sulfopropylated (SPEI) derivatives were successfully and homogenously attached onto GO, as confirmed by various characterization techniques, yielding GO-PEI, and novel GO-GPEI and GO-SPEI nanohybrids. The antibacterial activity of these nanohybrids was assessed against Gram (-) Escherichia coli and Gram (+) Staphylococcus Aureus bacteria. Both GO-GPEI and GO-SPEI were found to exhibit higher antibacterial activity, specifically against E. coli bacteria, compared to the pristine GO and GO-PEI nanohybrid, with GO-SPEI being more active than GO-GPEI. Finally, GO-GPEI and GO-SPEI were found to exhibit low cytotoxicity against mammalian cells, signifying that they can be used as potential antibacterial agents in various applications, including those in the disinfection industry.