As a widely studied photoactive antibacterial nanomaterial, the intrinsic antibacterial traits of graphitic carbon nitride (g-C3N4) as a two-dimensional nanomaterial have not been reported so far. Herein, nitrogen-plasma-treated g-C3N4 (N-g-C3N4) nanosheets and their influence on bactericidal characteristics are investigated. Bactericidal rates of more than 99% have been successfully achieved for 8 kinds of foodborne pathogenic bacteria by N-g-C3N4 with 8 h incubation in the dark. The achieved rates are percentage wise 10 times higher than those for pristine g-C3N4. Cell rupture caused by direct mechanical contact between g-C3N4 nanosheets and cell membranes is observed. X-ray photoelectron spectroscopy revealed a substantial loss of surface defects and nitrogen vacancies in N-g-C3N4. Molecular dynamics simulations further indicated that the largely sealed defects of N-g-C3N4 enhanced the electrostatic attraction between inherent pores and lipid heads; thus, further insertion of N-g-C3N4 was promoted, resulting in enhanced antibacterial activity. This study establishes novel fabrication and application strategies for carbon based antibacterial nanomaterials.