Background Human metapneumovirus (hMPV) is a major respiratory viral pathogen in young children, elderly individuals and immunocompromised patients. Despite its major effects related to bronchiolitis, pneumonia and its potential role in recurrent wheezing episodes, there is still no commercial treatment or vaccine available against this paramyxovirus. Methods We tested a therapeutic strategy for hMPV that was based on RNA interference. Results An hMPV genome-wide search for small interfering RNAs (siRNAs) by computational analysis revealed 200 potentially effective 21-mer siRNAs. Initial screening with a luciferase assay identified 57 siRNAs of interest. Further evaluation of their inhibitory potential against the four hMPV subgroups by quantitative real-time reverse transcriptase PCR and plaque immunoassay identified two highly potent siRNAs with 50% inhibitory concentration (IC50) values in the subnanomolar range. siRNA45 targets the nucleoprotein messenger RNA (mRNA) and had IC50 values <0.078 nM against representative strains from the four hMPV subgroups, whereas siRNA60, which targets the phosphoprotein mRNA, had IC50 values between 0.090– <0.078 nM against the same panel of hMPV strains. Longer 25/27-mer siRNAs known as Dicer substrates designed from the top two siRNA candidates were also evaluated and were at least as effective as their corresponding 21-mer siRNAs. Interestingly, the presence of one or two nucleotide mismatches in the target mRNA sequence of some hMPV subgroups did not always affect hMPV inhibition in vitro. Conclusions We successfully identified two highly efficient siRNAs against hMPV targeting essential components of the hMPV replication complex.