The development of drug-resistant bacteria has become a public health problem, among which methicillin-resistant Staphylococcus aureus (MRSA) leads to various life-threatening diseases. Graphene oxide (GO) is a two-dimensional nanomaterial with potential in the anti-MRSA treatment. This study prepared GO nanosheets with fixed lamellar size, investigated its antibacterial activity against MRSA, and analyzed the related antibacterial mechanisms. We found that the fabrication of GO with stable dispersion was workable. Furthermore, such GO had superior antibacterial performance against MRSA at low concentrations with the dose-dependent anti-MRSA effect. The GO-MRSA interaction also provided fundamental support for the antibacterial mechanisms with cleavage and encapsulation effects. In conclusion, GO nanosheets may be a promising antimicrobial agent against MRSA.