RNA interference triggered by small interfering RNAs (siRNAs) can be used to effectively contain viral spread. Here, we report on the mechanism of action of siRNAs targeting the medically important coxsackievirus B3 (CVB-3) as a typical representative of viruses with a non-segmented RNA genome in positive-strand orientation. Antiviral siRNAs can be designed to target the genomic (+)-strand, the (−)-strand that occurs as a replication intermediate, or both. In the present study, two complementary and systematic approaches are presented providing direct evidence that silencing of the viral (+)-strand is the key to inhibit CVB-3: first, we used rational siRNA design to direct silencing activity specifically against either of the two viral strands. As a second approach, we employed siRNA containing modified nucleotides to render them specific for one of the virus RNAs. Experiments with infectious coxsackievirus revealed that the inhibitory efficiency correlates exclusively with the activity of the siRNAs directed against the viral (+)-strand. Our finding that only (+)-strand specific siRNAs exert significant antiviral potency may hold true for other RNA viruses with (+)-stranded genomes as well and may therefore be helpful in the development of efficient strategies to inhibit virus propagation.