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Objectives: To study how the semen microbiome profile in men with nonobstructive azoospermia (NOA) differs from that of fertile controls (FCs)., Design: Using quantitative polymerase chain reaction and 16S ribosomal RNA, we sequenced semen samples from men with NOA (follicle-stimulating hormone >10 IU/mL, testis volume <10 mL) and FCs and performed a comprehensive taxonomic microbiome analysis., Setting: All patients were identified during evaluation at the outpatient male andrology clinic at the University of Miami., Patients: In total, 33 adult men, including 14 diagnosed with NOA and 19 with proven paternity undergoing vasectomy, were enrolled., Main Outcome Measures: Bacterial species in the semen microbiome were identified., Results: Alpha-diversity was similar between the groups, suggesting similar diversity within samples, whereas beta-diversity was different, suggesting differences in taxa between samples. In the NOA men, the phyla Proteobacteria and Firmicutes were underrepresented, and Actinobacteriota were overrepresented compared with FC men. At the genus level, Enterococcus was the most common amplicon sequence variant in both groups, whereas 5 genera differed significantly between the groups, including Escherichia and Shigella, Sneathia, and Raoutella., Conclusion: Our study showed significant differences in the seminal microbiome between men with NOA and fertile men. These results suggest a loss of functional symbiosis may be associated with NOA. Further research into the characterization and clinical utility of the semen microbiome and its causal role in male infertility is necessary., (Copyright © 2023 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)