Enterovirus 71 (EV71) is one of the major etiological agents of human hand-foot-and-mouth disease (HFMD) worldwide. EV71 infection in young children and people with immunodeficiency causes severe symptoms with a high fatality rates. However, there is still no approved drugs to treat such infections. Based on our previous report of a peptide-aldehyde anti-EV71 protease, we present here a highly specific α-hydroxy-nitrile derivative NK-1.9k, which inhibited the proliferation of multiple EV71 strains and coxsackievirus A16 (CVA16) in various cells with EC50 of 37.0 nM with low cytotoxicity (CC50 > 200 μM). The hydroxy-nitrile covalent warhead conferred NK-1.9k high potency and selectivity to interact with the cysteine residue of the active site of the viral protease. We also documented the resistance to NK-1.9k with a N69S mutation in EV71 3Cpro. The combination of NK-1.9k and EV71 polymerase or entry inhibitors produced strong synergistic antiviral effects. Collectively, our findings suggest our compounds can potentially be developed as drugs for the treatment of HFMD.