In a first-of-its-kind longitudinal One Health Scottish study, environmental (soil, wastewater), livestock faeces and human samples (faecal, saliva, blood) will be collected in a two-year integrated approach. The aim is to assess the diversity, and distribution of taxa and microbial resistance within distinct ecological niches, as well as track microbial transmission and resistance dynamics across them. Shotgun metagenomic sequencing is a powerful untargeted sequencing approach that assays all microorganisms inhabiting an environmental niche. Multiple wet- and dry-lab methodological approaches have been developed to process shotgun data, aiming for high-fidelity reporting of true microbial diversity, as well as accurate taxa identification, taxa quantification, and genome assembly reconstruction. An array of DNA extraction methods have been developed for various sample types, and numerous bioinformatic pipelines have been developed for taxonomic profiling and metagenome-assembled genomes (MAG) reconstruction. Here, we benchmark key wet- and dry-lab processes, including DNA extraction protocols, taxonomic profiling, and MAG binning tools, to maximize capturing unbiased microbial diversity, accurate relative abundance estimation, and retrieve near-complete MAGs across our sampled One Health ecological niches. These key wet- and dry-lab benchmarking exercises have enabled us to construct an ecological niche-informed methodological framework, encompassing DNA extraction protocols, taxonomic profiling pipelines, and MAG generation pipelines, for analyzing the data generated in our One Health Scottish Study.