Non-toxic carbon-based hybrid nanomaterials based on carbon nanodisks were synthesized and assessed as novel antibacterial agents. Specifically, acid-treated carbon nanodisks (oxCNDs), as a safe alternative material to graphene oxide, interacted through covalent and non-covalent bonding with guanidinylated hyperbranched polyethyleneimine derivatives (GPEI5K and GPEI25K), affording the oxCNDs@GPEI5K and oxCNDs@GPEI25K hybrids. Their physico-chemical characterization confirmed the successful and homogenous attachment of GPEIs on the surface of oxCNDs, which, due to the presence of guanidinium groups, offered them improved aqueous stability. Moreover, the antibacterial activity of oxCNDs@GPEIs was evaluated against Gram-negative E. coli and Gram-positive S. aureus bacteria. It was found that both hybrids exhibited enhanced antibacterial activity, with oxCNDs@GPEI5K being more active than oxCNDs@GPEI25K. Their MIC and MBC values were found to be much lower than those of oxCNDs, revealing that the GPEI attachment endowed the hybrids with enhanced antibacterial properties. These improved properties were attributed to the polycationic character of the oxCNDs@GPEIs, which enables effective interaction with the bacterial cytoplasmic membrane and cell walls, leading to cell envelope damage, and eventually cell lysis. Finally, oxCNDs@GPEIs showed minimal cytotoxicity on mammalian cells, indicating that these hybrid nanomaterials have great potential to be used as safe and efficient antibacterial agents.