<h4>Background</h4>Microbiome research has expanded rapidly, however, lack of standardized and validated protocols for microbiome sampling and DNA extraction has hindered the reproducibility and comparability of studies. The SUS-MIRRI.IT project aimed to prepare and validate Standard Operating Procedures (SOPs) for microbiome analysis across diverse ecosystems, including fermented foods, soils, waters, and more. To validate these protocols, 15 Italian research units (RUs) participated in an interlaboratory trial on 120 samples (liquid and solid fermented foods, waters, and soils). Metataxonomic sequencing was performed using 16S rRNA gene amplicon sequencing to assess the reproducibility of the protocols. The interlaboratory trial involved distributing homogenized samples to participating RUs and evaluating performance both between and within RUs. This was done by comparing results obtained from DNA extraction and amplicon-based sequencing.<h4>Results</h4>The results demonstrated high reproducibility of the procedures suggested in the SOPs across different sample types, with no significant differences in microbial diversity or composition between biological replicates or research units. DNA recovery was generally consistent, with minor variations observed in solid samples.<h4>Conclusions</h4>This study underlines the importance of standardized protocols in microbiome research. The validated Standard Operating Procedures developed by the SUS-MIRRI.IT project demonstrate robustness and reproducibility across diverse ecosystems, providing a foundation for future microbiome studies. The adoption of these protocols will enhance data comparability and support large-scale meta-analyses in food systems microbiome research.