The aim of this study was to determine the bactericidal potential of graphene oxide (GO) in contact with four species of bacteria:E. coli,S. mutans,S. aureusandE. faecalis. Bacterial cell suspensions of each species were incubated in a medium containing GO, with incubation times of 5, 10, 30 and 60 min, at final concentrations of 50, 100, 200, 300 and 500 μg/mL. The cytotoxicity of GO was evaluated using live/dead staining. The results were recorded using a BD Accuri C6 flow cytofluorimeter. Obtained data were analyzed using BD CSampler software. A significant bacteria viability reduction was noted in all GO-containing samples. The antibacterial properties of GO were strongly influenced by GO concentration and incubation time. The highest bactericidal activity was observed at concentrations of 300 and 500 μg/mL for all incubation times (5, 10, 30 and 60 min). The highest antimicrobial potential was observed forE. coli: after 60 min, the mortality rate was 94% at 300 µg/mL GO and 96% at 500 µg/mL GO; the lowest was found forS. aureus—49% (300 µg/mL) and 55% (500 µg/mL).