We investigated the prevalence of various genital organisms in 268 men with (cases) and 237 men without (controls) urethral symptoms/signs (urethral discharge, dysuria and/or urethral irritation) from two sexual health clinics in Sydney between April 2006 and November 2007. The presence of urethral symptoms/signs was defined as non-gonococcal urethritis (NGU) for this study. Specific aims were to investigate the role of Ureaplasma urealyticum in NGU and the prevalence of Mycoplasma genitalium in our population. Multiplex polymerase chain reaction-based reverse line blot (mPCR/RLB) assay was performed to detect 14 recognized or putative genital pathogens, including Chlamydia trachomatis, M. genitalium, U. urealyticum and U. parvum. U. urealyticum was associated with NGU in men without another urethral pathogen (odds ratio [OR] 2.0, 95% confidence interval [CI] 1.1-3.8; P = 0.04); this association remained after controlling for potential confounding by age and history of unprotected vaginal sex in the last four weeks (OR 2.0, 95% CI: 1.1-3.9; P = 0.03). C. trachomatis (OR 7.5, P < 0.001) and M. genitalium (OR 5.5, P = 0.027) were significantly associated with NGU. The prevalence of M. genitalium was low (4.5% cases, 0.8% controls). U. urealyticum is independently associated with NGU in men without other recognized urethral pathogens. Further research should investigate the role of U. urealyticum subtypes among heterosexual men with NGU.