OBJECTIVE Acute epididymitis is often associated with urethritis. Mycoplasma genitalium and Ureaplasma urealyticum have been considered as pathogens of urethritis. The aim of the present study was to determine the prevalence of these microorganisms in men with acute epididymitis. METHODS A total of 56 men younger than 40 years-of-age with acute epididymitis were enrolled in the present study between January 2006 and June 2010. First-void urine specimens were subjected to culture of aerobic bacterial species, and examined for the presence of Chlamydia trachomatis, M. genitalium, M. hominis, U. parvum and U. urealyticum by polymerase chain reaction-based assays. Urethral swabs were cultured for Neisseria gonorrhoeae. RESULTS The number and percentage of patients positive for each microorganism were as follows: Gram-negative bacilli, 2% and 3.6%; Gram-positive cocci, 23% and 41.1%; N. gonorrhoeae, 3% and 5.4%; C. trachomatis, 28% and 50.0%; M. genitalium, 5% and 8.9%; M. hominis, 6% and 10.7%; U. parvum, 6% and 10.7%; and U. urealyticum, 5% and 8.9%. Among 25 men with non-chlamydial non-gonococcal epididymitis, who were negative for Gram-negative bacilli, M. genitalium or U. urealyticum was detected in one man each (4.0%), and M. hominis and/or U. parvum was detected in five (20.0%). CONCLUSIONS In men younger than 40 years-of-age with acute epididymitis, C. trachomatis is a major pathogen. The prevalence of genital mycoplasmas and ureaplasmas are lower, and the role of genital mycoplasmas and ureaplasmas in the development of acute epididymitis remains to be determined.