Since the first description of a commensal seminal microbiome using sequencing, less than a decade ago, interest in the composition of this microbiome and its relationship with fertility has been growing. Articles using next-generation sequencing techniques agree on the identification of the most abundant bacterial phyla. However, at the genus level, there is still no consensus on which bacteria are most abundant in human seminal plasma. This discrepancy may be due to methodological variability such as sample collection, bacterial DNA extraction methodology, which hypervariable regions of16S rRNAgene have been amplified, or bioinformatic analysis. In the present work, seminal microbiota of 14 control samples and 42 samples of idiopathic infertile patients were characterized based on full-length sequencing of the16S rRNAgene using MinION platform from Oxford Nanopore. These same samples had been analyzed previously using Illumina’s MiSeq sequencing platform. Comparison between the results obtained with the two platforms has been used to analyze the impact of sequencing method on the study of the seminal microbiome’s composition. Seminal microbiota observed with MinION were mainly composed of the phylaFirmicutes,Proteobacteria,BacteroidetesandActinobacteria, with the most abundant genera beingPeptoniphilus,Finegoldia,Staphylococcus,Anaerococcus,Campylobacter,Prevotella,Streptococcus,Lactobacillus,EzakiellaandEnterococcus. This composition was similar to that found by the Illumina platform, since these 10 most abundant genera were also among the most abundant genera detected by the Nanopore platform. In both cases, the top 10 genera represented more than 70% of the classified reads. However, relative abundance of each bacterium did not correlate between these two platforms, with intraindividual variations of up to 50 percentage points in some cases. Results suggest that the effect of the sequencing platform on the characterization of seminal microbiota is not very large at the phylum level, with slightly variances inFirmicutesandActinobacteria, but presents differences at the genus level. These differences could alter the composition and diversity of bacterial profiles or posterior analyses. This indicates the importance of conducting multi-platform studies to better characterize seminal microbioma.