RNA interference (RNAi) has been shown to be suitable to inhibit viruses in experimental setups and is considered a promising antiviral strategy that is currently being tested in various clinical trials. The present study provides an approach to design siRNAs with high potency against a virus-specific target gene. In recent years, several outbreaks of aseptic meningitis caused by an echovirus 30 (EV-30) infection have been described. Based on an initial set of 30 in silico designed siRNAs, six siRNAs targeting the 3D RNA-dependent RNA-Polymerase (3D(Pol)) of EV-30 were selected. All but one of them showed high efficiency in both, reporter and virus assays. A second aim of the study was to re-investigate the relevance of the decay-accelerating factor (DAF, also known as CD55) as cellular entry receptor of EV-30 by means of RNAi, a question which had been under debate in previous studies. Knockdown of DAF inhibited drastically infection by EV-30 indicating that DAF plays an important role either as an attachment factor or as a receptor.