We aimed to study the correlation between leukocyte counts in semen and bacterial pathogens in seminal samples of infertile men, and to establish the minimum leukocyte count associated with significant bacteriospermia. A total of 116 patients who underwent evaluation of fertility were investigated using routine semen analysis according to the guidelines of the WHO and bacterial pathogens analysis by culture and in-house PCR assay. The overall prevalence of bacteriospermia in semen samples was 56.9% independent of the presence of leukocytes. The most common bacterial species detected were Chlamydia trachomatis (41.4%), Ureaplasma urealyticum (15.5%) and Mycoplasma hominis (10.3%). The receiver operating characteristic curve analysis demonstrated that the sensitivity/specificity for detecting bacteria at a cut off level of >or=1 x 10(6) leukocytes per ml (which is the WHO defined level for leukocytospermia) was 20.3%/81.5%. The highest sensitivity/specificity ratio was found in semen samples with a cut-off level of >or=0.275 x 10(6) leukocytes per ml, which is best shown with the odds ratio of 2.47. A significant correlation was found between bacteriospermia and leukocytospermia at the cut-off level of >or=0.275 x 10(6) leukocytes per ml of semen samples (P = 0.032). We proposed that this is a possible new cut-off level to predict the presence of bacteria in semen of infertile men.