Hand, foot and mouth disease (HFMD) is a serious, highly contagious disease. HFMD caused by Enterovirus 71 (EV71), results in severe complications and even death. The pivotal role of EV71 3Cpro in the viral life cycle makes it an attractive target for drug discovery and development to treat HFMD. In this study, we identified novel EV71 3Cpro inhibitors by docking-based virtual screening. Totally 50 compounds were selected to test their inhibitory activity against EV71 3Cpro. The best inhibitor DC07090 exhibited the inhibition potency with an IC50 value of 21.72 ± 0.95 μM without apparent toxicity (CC50 > 200 μM). To explore structure-activity relationship of DC07090, 15 new derivatives were designed, synthesized and evaluated in vitro enzyme assay accordingly. Interestingly, four compounds showed inhibitory activities against EV71 3Cpro and only DC07090 inhibited EV71 replication with an EC50 value of 22.09 ± 1.07 μM. Enzyme inhibition kinetic experiments showed that the compound was a reversible and competitive inhibitor. The Ki value was determined to be 23.29 ± 12.08 μM. Further molecular docking, MD simulation and mutagenesis studies confirmed the binding mode of DC07090 and EV71 3Cpro. Besides, DC07090 could also inhibit coxsackievirus A16 (CVA16) replication with an EC50 value of 27.76 ± 0.88 μM. Therefore, DC07090 represents a new non-peptidyl small molecule inhibitor for further development of antiviral therapy against EV71 or other picornaviruses.