Aims: Bacterial vaginosis (BV) is characterized by a transition in vaginal microflora from lactobacilli to anaerobic bacteria. Gardnerella vaginalis and Atopobium vaginae are considered the most responsible pathogens for the etiology of BV. Colonization of male urethra with BV-associated bacteria has been rarely investigated. The aim of this study was to investigate the differences in the presence of BV-associated bacteria in the healthy male urethra in regard to sexual exposure. Methodology and results: The first-catch urine specimens, representative of urethral swabs, from 114 healthy male volunteers, were included in this study. Lactobacillus spp., L. crispatus, L. jensenii, L. gasseri, L. iners, G. vaginalis, A. vaginae, Peptoniphilus spp., P. lacrimalis, BVAB2, Mageeibacillus indolicus, Megasphaera type I, Mobiluncus mulieris, Leptotrichia/Sneathia, Corynebacterium spp., and Prevotella spp. were investigated using a PCR assay. The most frequently identified BV-associated bacteria were Lactobacillus spp., Peptoniphilus spp., and G. vaginalis. There was no association between any BV-associated bacteria and sexual exposure. There was statistically significant co-occurrence of A. vaginae and G. vaginalis in the MU of subjects independently of sexual exposure (p = 0.025). Also, there was a significant association between G. vaginalis and smoking (p = 0.023). Conclusion, significance and impact of study To the best of our knowledge, this is the first study reporting the co-occurrence of G. vaginalis and A. vaginae in the male urethra independently of sexual exposure.