The objective was to explore presence/detection of microorganisms in the male reproductive tract (PMMRT) in asymptomatic patients undergoing infertility treatment and their effects on semen quality in our region. This study enrolled 205 men (mean age, 35.9 years) in a single-centre, tertiary university hospital from December 2015 to December 2016. We used the modified Meares-Stamey test, real-time polymerase chain reaction (rt-PCR) and the National Institutes of Health Chronic Prostatitis Sympton Index (NHI-CPSI) questionnaire to address this issue. No patient met the prostatitis criteria by the modified Meares-Stamey 4-sample test, 33 (16.1%) were positive for rt-PCR in the first-voided urine for any of the Mycoplasma (Ureaplasma urealyticum/parvum, Mycoplasma hominis/genitalium) and C. trachomatis was detected in two cases (1%), and three for rt-PCR in semen for HPV high-risk genotypes non-16/18 (1.5%). Significant statistical differences were reported among patients with and without PMMRT in terms of lower rate of progressive spermatozoa (PR) (p < .034), total motile sperm count (p < .028), normal morphologic forms, especially in the sperm head (p < .001) and highest viscosity (p < .012). It was concluded that PMMRT, specially Mycoplasmas, in asymptomatic infertility men, affects semen quality. The NIH-CPSI questionnaire was not a valid initial screening to subsequently evaluate the presence of prostatitis/PMMRT.