To select the optimal graphene oxide (GO) for anticaries dental applications, aqueous dispersions containing GO nanosheets with various oxygen-containing functional groups were prepared and characterized using atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy. The antibacterial effect towards Streptococcus mutans (S. mutans) in both planktonic and biofilm forms was studied by colony forming units (CFU) counting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay, live/dead fluorescent staining, and confocal laser scanning microscopy (CLSM) observation. The oxidation capacity of different GO nanosheets was examined by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay and in vitro glutathione (GSH) oxidation assay. The results indicated that GO exerted strong antibacterial activities in a concentration-dependent manner towards S. mutans in both planktonic and biofilm forms. The minimum bactericidal concentration (MBC) was 40 μg/mL for planktonic S. mutans. When the concentration was higher than 80 μg/mL, 80% of the bacteria in the biofilms were devitalized. GO nanosheets with more oxygen-containing functional groups exerted higher toxicity at low concentrations. The functional groups of GO played a crucial role in its antibacterial outcome.