Reliable evaluations of soil biodiversity represent a key factor in understanding ecosystem services. To date, species-discriminating barcodes efficiently describe bacterial and fungal communities associated with environmental samples, whereas investigations of soil microfauna are often hampered by the lack of a marker region encompassing the taxonomic range of soil organisms. Two new PCR primer sets targeting the V4-V5 and V5-V7 variable regions of the ribosomal 18S RNA (18S rRNA) were designed to be specific for metazoans metabarcoding and capable of detecting the majority of their lineages. In silico and in vivo assays on four soil typologies were carried out to compare the newly developed primer sets with a selection of primers targeting the homologous gene, which were previously used to assess soil metazoan biodiversity. The new primer sets, both on the basis of the in silico and in vivo comparisons, were very selective and consistent when analysing metazoan biodiversity across the tested soil typologies. On the basis of the coverage index and taxonomic resolution, the new primers targeting the ribosomal 18S RNA outperformed the other primers, and they represent a promising tool for assessing soil metazoan biodiversity through metabarcoding approaches.