The testicular environment is immunoprivileged to protect germ cells from autoimmune and anti-inflammatory activities, but, on the other hand, it is susceptible to pathogens. Until now, the works that investigated the microbiological diversity in this environment were restricted to analyzing specific bacteria or viral communities. In this study, we evaluated the diversity in the seminal human microbiome using a whole-genome sequencing approach in a seminal human pool. For this, we collected 50 samples donated by participants from a public reproductive health service in Brazil. We observed a high proportion of the Bacteria domain (71.3%), whose largest groups are Bacillus, Staphylococcus, Mycobacterium, and Streptococcus. The Eukaryotic domain (27.6%) comprises Plasmodium, Trypanosoma, and Trichinella. Viruses (1.1%) are composed of Gammaretrovirus, Herv-K, and Herv-W. These findings expand the current view of microbial diversity in human semen and point out that evaluating uncultivated pathogens could be crucial before concluding reproductive and prophylactic treatments. In addition, the Herv families identified in seminal samples deserve studies with a functional and evolutionary perspective. These data contribute to identifying potential pathogens present in the semen (gamma diversity) and their correlations and opening a new front for research in the diagnosis of fertility-related diseases.