Enteroviral diseases are widespread and impose significant importance in medicine. Although the outcome of diseases that are associated with enteroviruses such as myocarditis, pancreatitis, hepatitis, or encephalomyelitis might be fatal, no specific antiviral therapy is yet available. We and others have shown that RNA interference (RNAi) effectively limits picornaviral replication and cytopathogenicity and improves survival in susceptible mice. However, little is known about the dependence of short interfering RNA (siRNA) efficacy on target region selection and emergence of viral escape mutants that may limit the effect of RNAi. The results of our study indicate that antiviral siRNA should be targeted preferentially to nonstructural protein coding regions because siRNA efficacy was consistently found to be superior compared to noncoding or structural protein coding regions. Further more, emergence of viral escape mutants that harbor single point mutations in the central part of the siRNA binding motif are the major factor that limits early therapeutic siRNA efficacy. The appearance of viral escape mutants can be sufficiently suppressed by combined administration of at least three distinct siRNA molecules. Therefore, genomic target selection and viral escape mutants are the most critical factors that limit early RNAi directed against enteroviral genomes. Both obstacles can be circumvented by appropriate target selection and combined siRNA administration.