In recent decades, the study of the gut microbiota has gained attention as a crucial element in human health and the onset of diseases. Technological advancements, such as high-throughput sequencing, have enabled a deeper study of intestinal microbiota composition and functions. Among the environmental factors modulating the gut microbial configuration, the impact of nutrients is of particular significance. Nonetheless, research on dietary lipids, particularly omega-3 fatty acids, remains underexplored. This study aimed to contribute to the metagenomic analysis of the 16 subunit of the ribosomal RNA gene in faecal samples. A nutritional intervention was carried out focused on fish consumption rich in omega-3. Successful DNA extraction from faecal samples yielded notable, with defined genomic DNA bands in the gel electrophoresis and an adequate concentration. Polymerase chain reactions, essential for library preparation, revealed distinct products in the gel electrophoresis, confirming the successful amplification of the V3-V4 16 ribosomal RNA region with proper adapter addition for DNA sequencing. Comprehensive quality control assessment of sequenced samples confirmed the effectiveness of the refined laboratory procedures. The steps undertaken in this project were critical for subsequent bioinformatics and biostatistics analyses, providing insights into the impact of omega-3-rich fish consumption on the relative abundance and diversity of studied microbial taxa. Integrating metagenomics analysis with other omics and non-omics disciplines, alongside standardising methods across different laboratories, will pave the way for the future application and democratisation of precision nutrition to enhance overall wellbeing.