A non-syndromic approach to treatment of people with non-gonococcal urethritis (NGU) requires identification of pathogens and understanding of the role of those pathogens in causing disease. The most commonly detected and isolated micro-organisms in the male urethral tract are bacteria belonging to the family of Mycoplasmataceae, in particular Ureaplasma urealyticum and Ureaplasma parvum. To better understand the role of these Ureaplasma species in NGU, we have performed a prospective analysis of male patients voluntarily attending a drop in STI clinic in Oslo. Of 362 male patients who were tested for NGU using microscopy of urethral smears, we found the following sexually transmissible micro-organisms: 16% Chlamydia trachomatis, 5% Mycoplasma genitalium, 14% U. urealyticum, 14% U. parvum and 5% Mycoplasma hominis. We found a high concordance in detecting in turn U. urealyticum and U. parvum using 16s rRNA gene and ureD gene as targets for nucleic acid amplification testing (NAAT). Whilst there was a strong association between microscopic signs of NGU and C. trachomatis infection, association of M. genitalium and U. urealyticum infections in turn were found only in patients with severe NGU (>30 polymorphonuclear leucocytes, PMNL/high powered fields, HPF). U. parvum was found to colonise a high percentage of patients with no or mild signs of NGU (0–9 PMNL/HPF). We conclude that urethral inflammatory response to ureaplasmas is less severe than to C. trachomatis and M. genitalium in most patients and that testing and treatment of ureaplasma-positive patients should only be considered when other STIs have been ruled out.