Background: Previous studies have relied on culture-dependent methods to determine microbial communities that may be found in the seminal fluids of men seeking reproductive health care. However, understanding the microbiome composition present in seminal fluids with the state-of-art next-generation sequencing technology is more germane than ever before, instead of culture methods which fails to identify over 99% of bacterial organisms present in biological samples. Methods: Forty semen samples were collected and after bacterial DNA extraction, 22 samples that passed quality check were used for amplification of the V4 region of the 16S rRNA using custom bar‑coded primers prior to sequencing with Illumina NextSeq 500 platform. Sequencing was performed in a pair-end modality rendering 2 x 150 basepair sequences. Sequence reads were imported into Illumina BaseSpace Metagenomics pipeline for 16S rRNA recognition. Distribution of taxonomic categories at different levels of resolution was done using Greengenes database. Results: The taxonomic categories from the dataset produced phyla that ranges from 6 to 25; Class (9-49), Order (16-99), Family (42-214), Genus (55-555) and Species (56-1156). The taxonomic profiles represented 25 phyla, showing 39.5% of the total sequence reads were categorized to Proteobacteria as the most abundant. This was followed by Firmicutes (33.54%), Actinobacteria (20.77%) and Bacteroidetes (4.77%), Fusobacteria (0.613%), Tenericutes (0.31%) and Verrucomicrobia (0.12. At the species taxonomic level 1841 species were identified among the seminal fluid samples. Serratia marcescens (23.61% sequence reads) was the most abundant species found in 9/22 of the samples followed by Lactobacillus iners (18.22%) 13/22, Serratia entomophila (5.54%) 17/22, Haemophilus parainfluenzae (3.64%) 10/22, Corynebacterium tuberculostearicum (3.29%) 21/22, Gardnerella vaginalis 2.39%) 12/22, Lactobacillus taiwanensis (2.08%) 10/22, Enterobacter amnigenus (1.63%) 15/22, Corynebacterium genitalium (1.29%) 12/22, Neisseria lactamica (1.18%) 8/22, Finegoldia magna (1.17%) 16/22, Prevotella bivia (1.14%) 10/22, Corynebacterium imitans (1.12%) 15/22, Corynebacterium jeikeium (1.02%) 17/22 and Lactobacillus acidophilus (1.01%) found in 6/22 of the samples. Conclusions: We investigated the microbiome compositions from seminal fluids and showed that there are varying bacterial diversities that are unique in each sample in contrast to culture-dependent methods.