Ureaplasma urealyticum and Mycoplasma hominis are most important genital mycoplasma responsible for many genitourinary tract infections. Nongonococcal urethritis (NGU) and bacterial prostatitis are most clinically significant infections that affect a wide spectrum of male age groups. The incorrect or inappropriate diagnosis and susceptibility testing leads to unsuitable medication and as a results delay of recovery and bad squeal like infertility. During a period of sex months, twenty two expressed prostate secretion (EPS) samples and eighteen urethral swabs were collected from patients with age (44.55±21.22 years). All samples were inoculated to strip and after 24 hours at 37° C the results recorded. The results of detection of genital mycoplasma revealed that only 3 (16.7%) of expressed prostate secretion samples give positive results for both Ureaplasma urealyticum and Mycoplasma hominis while 10 (45.5%) urethral swab were positive for both Ureaplasma urealyticum and Mycoplasma hominis. Common age group of prostatitis infected with genital mycoplasma is (41-60 years) while urethritis was detected among all three groups. The results of antibiotic susceptibility revealed that all isolates were fully sensitive (100%) to all antibiotics incorporated within the well except for erythromycin, roxithromycin, tetracyclin and levofloxacin in which one isolate (7.7%) was exhibit resistance in both Ureaplasma urealyticum and Mycoplasma hominis. The current study conclude the genital mycoplasmas (Ureaplasma urealyticum and Mycoplasma hominis) are predominant among non gonococcal urethritis and the macrolides still the choice for treatment while prescription of quinolones and tetracyclin must be under control due to emergence of resistance isolates.