The microbial communities inhabiting the reproductive tract play an important role in reproductive health. During unprotected sexual intercourse, the microorganisms present in the semen and vagina of heterosexual couples interact closely with each other. The concept of the "seminovaginal microbiome" has recently emerged to refer to all microorganisms present in both environments. Each partner influences the microbial composition of the other, even sharing several bacterial taxa. Therefore, to improve the understanding of this interaction, we aimed to analyse the microbial composition of semen and vagina samples from heterosexual couples and to assess their similarity in terms of composition and diversity. The selected cohort consisted of 50 infertile couples before entering infertility treatment from whom semen and vaginal samples were collected. Bacterial genomic material was extracted from the samples, followed by amplification and sequencing of the V4 hypervariable region of the 16S ribosomal RNA (rRNA) gene to allow for the taxonomic assignment of the microorganisms at the genus level. A total of 443 bacterial genera were identified in the semen samples and 107 genera in the vaginal samples. α-diversity analysis revealed that the semen samples had higher bacterial diversity and richness than the vaginal samples. In addition, ß-diversity analysis showed significant dissimilarities in bacterial genus composition between the semen and vaginal samples of the couples. However, 39 bacterial genera were shared by both niches, supporting an active exchange of microorganisms during unprotected sexual intercourse. Focusing research on couples' microbiomes from a holistic view may provide a better understanding of their interactions, which could help to develop more effective strategies to improve reproductive success.