Popularization of the real-time polymerase chain reaction method (RT-PCR), which is a trend of the recent years, allowed to significantly expand of the range of microorganisms that can be detected in the genitourinary tract of men. Moreover, the available picture of the microbiome's bacterial component structure became more detailed. Lactobacillus spp. remains one of the least studied groups of microorganisms. Treating patients with reproductive disorders, the authors have accumulated clinical experience demonstrating the possible relationship between presence of Lactobacillus spp. in the ejaculate and changes in the level of sex hormones and the key values registered with a spermogram. This study aimed to compare the levels of luteinizing hormone, follicle-stimulating hormone, testosterone, estradiol, prolactin, progesterone, and sex hormone binding globulin (SHBG) in blood serum and changes in spermogram values in 210 men with and without Lactobacillus spp. detected in their ejaculate. The treatment group included 105 men whose ejaculate had Lactobacillus spp. in the amount of (Lg) ≥ 103, as detected by RT-PCR. The control group included 105 men whose ejaculate did not have Lactobacillus spp. detected; the microbiome's bacterial component structure of their ejaculate was normal. Compared to the control group, treatment group had hormonal disorders registered more often: abnormal levels of three or more hormones (p = 0.04), hyperestradiolemia (p = 0.05), increased level of SHBG (p = 0.01). It was established that the presence of Lactobacillus spp. in the ejaculate of treatment group participants is associated with oligoastenoteratozoospermia (p < 0.01), decreased concentration of spermatozoa (p = 0.01), their decreased motility (p < 0.01) morphology abnormalities (p < 0.01). Thus, the presence of Lactobacillus spp. in the ejaculate can be interpreted as an additional marker of hormonal imbalance and fertility dysfunction in men.