The rising incidence of drug resistant diseases has led to an increasing need for developing novel and efficient antimicrobial products that can counter these infections. We report for the first time, the exceptional antibacterial activity of N-doped carbon nanosheets (CNS). The antibacterial activity and mechanism of action of CNS was examined for gram negative E. coli. Based on the cell viability tests, nucleic acid quantitation, time and concentration dependent antibacterial activity tests and SEM and TEM micrographs, performed under similar concentration and incubation conditions, the CNS dispersion shows the highest antibacterial activity, sequentially followed by GO, rGO and CCM, with a loss of cell viability by 92.1 ± 1.7%. We envision that the physical stress and piercing action caused by sharp "knife-edges" as well as the presence of heteroatoms in CNS result in the rupturing of the bacterial cell wall, eventually causing cell death. The high ID/IG ratio (0.99) of CNS is closely related to the formation of structural and edge plane defects, especially in the case of N-doped carbonaceous materials, which is one of the key factors in enhancing the antibacterial activity of the material.