Environmental DNA (eDNA) metabarcoding is emerging as a novel tool for monitoring soil biodiversity. Soil biodiversity, critical for soil health and ecosystem services, is currently under-monitored due to the lack of standardized and efficient methods. We assessed whether refinements to sampling and molecular protocols could improve soil biodiversity detection and monitoring. Comparing the 2018 LUCAS soil biodiversity protocols with newly developed national methods, we tested sampling topsoil (0-10 cm) versus deeper layers, larger soil sample sizes for DNA-extraction, taking more subsamples for composite soil samples, and alternative primer sets across 9 Belgian Biopoints included in the LUCAS 2022 survey. The results suggest that significantly more species can be detected in upper soil layers, including the forest floor, while the diversity of taxa and eDNA in the 10–30 cm soil layer is insufficient for annelids and arthropods to serve as indicators of ecological change. Additionally, comparison of the universal eukaryotic primers (18S) with primer sets tailored to soil mesofauna and macrofauna, showed that universal 18S primers provide limited resolution for Collembola and Annelida. Overall, the analyses suggest that vertical soil stratification (with two sampling depths) has a greater influence on the captured diversity of soil mesofauna and macrofauna than the number of subsamples, and that the highest diversity is recovered when surface sampling (0–10 cm topsoil and forest floor) is combined with a greater number of subsamples and a larger sampled area. With refinement and standardization, eDNA metabarcoding, combined with optimized sampling protocols, could become a powerful and efficient tool for monitoring soil biodiversity in European soils. Description of the files This dataset includes interactive Krona taxonomy charts to visually summarize the diversity and relative read abundance of detected taxa across sampling locations and protocols. Each ring in the chart represents a taxonomic level, with the relative width of segments reflecting the proportion of reads assigned to specific taxa at that level. These charts enable exploration of taxonomic composition and allow for comparisons between the different sampled locations, sampling protocols tested, and primer sets tested. All krona charts were made in R using psadd::plot_krona. To correct for uneven sequencing depth per sample, datasets were rarefied using a random subsampling method to 27913, 31655, 1856, 19728, and 19632 reads for Annelida (Olig01), Collembola (Coll01), Fungi (ITS9mun/ITS4ngsUni), protists (18S), and Archaea (SSU1ArF/SSU1000ArR) respectively. Fauna datasets that are subsets of the total data recovered by a primer set designed to target many different phyla (e.g. 18S) were not rarefied prior to generating the krona plots. ejp_soil_annelida_olig01_27913.html contains the interactive taxonomy charts for Annelida. The data was generated using the group-specific Olig01 primer set and rarefied to 27,913 reads per sample. ejp_soil_collembola_coll01_31655.html contains the interactive taxonomy charts for Collembola. The data was generated using the group-specific Coll01 primer set and rarefied to 31,655 reads per sample. ejp_soil_arthropoda_inse01.html contains the interactive taxonomy charts for Arthropoda (Insecta, Arachnida, Chilopoda, Diplura, and Malacostraca). The data was generated using the Inse01 primer set. ejp_soil_fungi_its9mun_its4ngsuni_1856.html contains the interactive taxonomy charts for Fungi. The data was generated using the ITS9mun and ITS4ngsUni primer set and rarefied to 1,856 reads per sample. ejp_soil_protists_18s_19728.html contains the interactive taxonomy charts for protists. The data was generated using the eukaryotic 18S primer set and rarefied to 19,728 reads per sample. ejp_soil_archaea_ssu1arf_ssu1000arr_19632.html contains the interactive taxonomy charts for Archaea. The data was generated using the SSU1ArF and SSU1000ArR primer set and rarefied to 19,632 reads per sample. ejp_soil_annelida_18s.html contains the interactive taxonomy charts for Annelida. The data was generated using the eukaryotic 18S primer set. ejp_soil_collembola_18s.html contains the interactive taxonomy charts for Collembola. The data was generated using the eukaryotic 18S primer set. ejp_soil_arthropoda_18s.html contains the interactive taxonomy charts for Arthropoda. The data was generated using the eukaryotic 18S primer set. ejp_soil_metadata.csv contains metadata for the samples in this study. It includes information about the sampling locations, the sampling protocols used, the sampling depth (cm), land use type, EUNIS habitat classification, and the LUCAS-ID for each sample.