Graphene oxide and its derivatives have been widely explored for their antimicrobial properties due to their high surface-to-volume ratios and unique chemical and physical properties. However, little information is available on their effects on viruses. In this study, we report the broad-spectrum antiviral activity of GO against pseudorabies virus (PRV, a DNA virus) and porcine epidemic diarrhea virus (PEDV, an RNA virus). Our results showed that GO significantly suppressed the infection of PRV and PEDV for a 2 log reduction in virus titers at noncytotoxic concentrations. The potent antiviral activity of both GO and rGO can be attributed to the unique single-layer structure and negative charge. First, GO exhibited potent antiviral activity when conjugated with PVP, a nonionic polymer, but not when conjugated with PDDA, a cationic polymer. Additionally, the precursors Gt and GtO showed much weaker antiviral activity than monolayer GO and rGO, suggesting that the nanosheet structure is important for antiviral properties. Furthermore, GO inactivated both viruses by structural destruction prior to viral entry. The overall results suggest the potential of graphene oxide as a novel promising antiviral agent with a broad and potent antiviral activity.