A broad spectrum of bacteria, fungi, protozoa and viruses can cause urethritis. In particular, N. gonorrhoeae, C. trachomatis, M. genitalium and T. vaginalis are the focus of diagnostic considerations as classic pathogens associated with sexually transmitted infections (STI). A step-by-step procedure is needed to make a definitive diagnosis. Microscopy with a staining preparation provides an initial differentiation between gonoccocal and non-gonococcal urethritis in symptomatic men as a point-of-care (POC) test. Nucleic acid amplification technology (NAAT) is used for specific and sensitive pathogen detection and, as a multiplex diagnostic test, offers the possibility of detecting several organisms from the same sample. In addition, compared to culture, no vital organisms are required, which allows the collection and use of more diverse and less invasive biological samples (e.g. first stream urine in men or vaginal swabs). Susceptibility testing by culture remains essential for N. gonorrhoeae as resistance is emerging. The treatment of urethritis depends on the suspected or proven pathogen according to the current guidelines. Treatment failure can be caused by many factors (coinfection, lack of therapy adherence, reinfection or resistance of the pathogen) and requires a repeated diagnostic and therapeutic procedure and differentiated approach.