Growing resistance of bacteria towards antibiotics resulted in extensive research effort for development and application of new materials and techniques. Due to their unique properties, graphene quantum dots (GQDs) have attracted much attention and are a promising material with potential applications in many fields. One use of GQDs is as a photodynamic therapy agent that generates singlet oxygen. In this work, GQDs synthesized by focusing nanosecond laser pulses into a mixture of benzene and nickel(II) oxide were combined with methylene blue (MB) to eradicate Gram-negative Escherichia coli and Gram-positive Micrococcus luteus. Theoretical calculation of pressure evolution was calculated using the standard finite difference method. Detailed characterizations were performed with transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier-transform infrared (FTIR), UV-Visible (UV-Vis), and photoluminescence (PL) spectra. Furthermore, singlet oxygen generation from MB-GQD mixture was investigated by measuring the rate of 9,10-anthracenediyl-bis(methylene) dimalonic acid photobleaching at 400 nm. Combining MB with GQDs caused enhanced singlet oxygen generation, leading to improved bacterial deactivation rate. The (3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide) (MTT) assay was used to determine if GQDs in dark conditions caused human cellular side-effects and affected cancer and noncancer cellular viability. We found that even high concentrations of GQDs do not alter viability under dark conditions. These results suggest that the MB-GQD combination is a promising photodynamic therapy agent that may be useful when antibiotics resistance is present.