In this paper, we report on the antibacterial activity of graphene oxide (GO) nanosheets synthesized by the modified Hummers method. The as-prepared GO nanosheets were characterized using UV-vis spectroscopy, XRD, FT-IR spectroscopy, XPS, TEM and Raman spectroscopy analysis. The antibacterial activity of GO nanosheets towards Gram-negative bacteria, “Escherichia coli” and Gram-positive bacteria, “Streptococcus iniae,” was investigated by the colony counting method. The obtained results indicated that GO nanosheets were more effective towards Gram-positive bacteria. The mechanism underlying the antibacterial behavior of GO nanosheets involved the generation of reactive oxygen species, which was confirmed using the electron spin resonance technique coupled with spin trapping technique. These results proved that GO nanosheets can be used as a potential antibacterial agent in future applications of nanomedicine.