In the present study, chitosan-containing nanocomposites were investigated as new antibacterial agents. Magnetite (Fe3O4) nanoparticles (NPs) as well as chitosan (CS)/Fe3O4 nanocomposites (NCs) and graphene(Gr)/ CS/Fe3O4 NCs were synthesized by simple hydrothermal method. Their composition, structure and morphology were studied, followed by the evaluation of their antibacterial activity against ESBL-producing and gramnegative P. aeruginosa and K. pneumoniae bacterial strains. The Gr/CS/Fe3O4 NCs showed significantly higher antibacterial activity compared to Fe3O4 NPs and CS/Fe3O4 NCs (105 and 69 % higher against P. aeruginosa as well as 91 and 77 % higher against K. pneumoniae, respectively). The minimum inhibitory concentration (MIC) of Gr/CS/Fe3O4 NCs against P. aeruginosa and K. pneumoniae were 60 and 70 mu g/mL, respectively. The synergistic antibacterial activity and facile synthesis of Gr/CS/Fe3O4 NCs suggests their applicability as novel highly efficient antibacterial agents with potential for a wide range of biomedical applications, where antibacterial properties are needed.