Urease-positive urogenital mycoplasmas are considered to be responsible for the formation of urinary stones. They are usually a part of the normal flora in the human urogenital tract, causing asymptomatic infections. However, many symptomatic infections with these bacteria have been reported. M. genitalium is recognized as a cause of male urethritis and other common genitourinary diseases. The role of other urogenital mycoplasmas is still unclear. The aim of this study was to estimate the quantitative prevalence of Ureaplasma spp., M. genitalium and M. hominis in men with urolithiasis using quantitative real-time PCR (qPCR). The study group comprised 100 men with urolithiasis. A total of 60 men were included in the control group. Urogenital mycoplasma DNA in urine samples was detected significantly more often among men with urolithiasis than in healthy subjects—43.0% vs. 26.6%, p = 0.0382, respectively. The majority of positive results (38/43) concerned U. parvum species, the frequency of which was higher in the study group (38.0% (38/100)) than in the control group (23.3% (14/60)), p = 0.0552. The median concentration of U. urealyticum DNA was higher in the study group compared with the control, p = 0.5714. However, further studies are needed to confirm the usefulness of quantitative studies in determining the role of urogenital mycoplasmas in pathology.