Targeted metagenomics is the solution of choice to reveal differential microbial profiles (defined by richness, diversity and composition) as part of case-control studies. It is well documented that each data processing step may have the potential to introduce bias in the results. However, selecting a bioinformatics pipeline to analyze high-throughput sequencing data from A to Z remains one of the critical considerations in a case-control microbiota study design. Consequently, the aim of this study was to assess whether the same biological conclusions regarding human gut microbiota composition and diversity could be reached using different bioinformatics pipelines. In this work, we considered four pipelines (mothur, QIIME, kraken and CLARK) with different versions and databases, and examined their impact on the outcome of metagenetic analysis of Ion Torrent 16S sequencing data. We re-analyzed a case-control study evaluating the impact of the colonization of the intestinal protozoa Blastocystis sp. on the human gut microbial profile. Although most pipelines reported the same trends in this case-control study, we demonstrated how the use of different pipelines affects the biological conclusions that can be drawn. Targeted metagenomics must therefore rather be considered as a profiling tool to obtain a broad sense of the variations of the microbiota, rather than an accurate identification tool.