As a powerful and novel nanocarrier, graphene oxide (GO) is employed to load a water insoluble antibacterial drug, gramicidin (GD), for effective antibacterial treatments. The loaded amount of GD on the surface of GO was calculated and was found to be 14% (wt%). The antibacterial activity of GO modified GD (GOGD) was measured against Pseudomonas aeruginosa and Staphylococcus aureus using plate counting, optical density (OD600), transmission electron microscopy (TEM), fluorescence (2D, 3D) and matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The use of multiple analytical approaches adds certainty to the cytotoxicity assessments of GOGD, which shows better efficiency than GO and GD. GOGD has potential wide-ranging effects against different bacterial strains. Nano-cytotoxicity mechanism was discussed in detail, and controversies in earlier results were refuted.