Among other things male sterility can be caused by inflammatory diseases of the urogenital tract, often associated with opportunistic microorganisms. Thus, it is necessary to implement modern methods for the detection and identification of opportunistic microorganisms in the urogenital tract. The aim of the work was to conduct comparative analysis of the ejaculate microbiota from men of the reproductive age and studied using quantitative polymerase chain reaction (PCR) and culture method. 86 samples of ejaculate collected from men aged 18–57 years after observing sexual abstinence for 3–5 days were examined. With culture study in 50% of samples we observed growth of gram positive facultative anaerobic bacteria in the amount less than 10³ CFU/ml; in 16.3% of samples — the growth of bacteria was not observed. With real-time PCR in each sample 8–15 groups of microorganisms were detected (including the prevailing groups) in the amount of 10²–10⁶ GE/ml. In all 86 samples obligate anaerobes that cannot not be cultured in vitro were detected. The predominant groups of microorganisms, as determined by real-time PCR, were detected by the culture method only in 24.4% of cases.