Background/Aim: This study aimed to determine the prevalence of Mycoplasma genitalium infection among male patients with dysuria and/or urethral discharge. An analysis of the clinical, demographic and microbiological factors associated with M.genitalium infection was also conducted. Method: From May 2007 to June 2011, men presenting to the clinic with self-reported symptoms of dysuria and/or urethral discharge were identified and underwent urethral swab, which was microscopically assessed for objective non-gonococcal urethritis. A first-void urine sample was tested for Chlamydia trachomatis and Neisseria gonorrhoeae using the Aptima Combo-2 assay. A portion of the urine sample was sent for polymerase chain reaction analysis for M.genitalium. Results: One thousand, one hundred and eighty-two men with dysuria and/or urethral discharge were tested for M.genitalium. Of those, 96 men (8.1%) were positive for M.genitalium. Men identifying as solely MSM (men who have sex with men) constituted 16.3% (n = 193) of the sample. Their infection rate was 3.1% (n = 6). The infection rate for heterosexual and bisexual men was 9.1%. For all men, the M.genitalium co-infection rate was 14.6% (n = 14) with C.trachomatis and 3.1% (n = 3) with N.gonorrhoeae. Factors associated with M.genitalium infection were analysed by univariate analysis. We determined that five investigated predictors were significantly associated with M.genitalium infection, urethral discharge, non-gonococcal urethritis on Gram stain of urethral smears, identification as heterosexual or bisexual, and absence of co-infection with C.trachomatis or N.gonorrhoeae. Conclusion: In Adelaide, M.genitalium is an important sexually transmitted infection among men with dysuria and/or urethral discharge, and is primarily an infection of heterosexual and bisexual men.