The aim of this study was to investigate how often etiology of chronic prostatitis syndrome is proved if the presence of the traditional prostatitis causitive pathogens is investigated with 'four glass test' plus the semen microbiological diagnostics. It also was to determine the frequency of traditional causitive pathogens of prostatitis in EPS and / or VB3 and the semen of patients with chronic bacterial prostatitis. The study included 107 adult male patients older than 20 years who came with chronic prostatitis to the 'Ambulance for urogenital infections and sexually transmitted disease' of the 'Clinics for Infectious Diseases' "Dr. Fran Mihaljevic" in the period from 2009 to 2013, and in whom the diagnosis of chronic bacterial prostatitis was confirmed by proving the causitive pathogens in EPS and / or VB3 and / or semen. The study was designed so that patients were control to each other. The experiment was carried out as a 'five glass test' in all subjects, and the analysis is done as it is in the same subjects performed the 'two glass test' and 'four glass test'. The most common traditional pathogens proven in EPS and / or VB3 and semen were Enterococcus faecalis (detected in 45 patients or 42.05%) and Escherichia coli (demonstrated in 42 patients or 39.25%). All other proven traditional pathogens - Proteus mirabilis, Klebsiella pneumoniae, Streptococcus agalactiae, coagulase - negative staphylococci - were isolated in EPS and / or VB3 and semen in 20 patients or 18.7%. Examining that samples it was determined that in 62 (57.94%) patients the pathogen was detected in EPS and / or VB3 and in the semen, in 18 (16.82%) patients the cause was found only in EPS and / or VB3, and in 27 (25.23%) patients the cause was found only in the semen. The etiology was confirmed in the significantly higher percentage of patients in EPS / VB3 and semen compared to the patients where etiology was demonstrated only in EPS / VB3 (Fischer exact test, p <0.00001), or only in the semen (Fischer exact test; p = 0.0012). There were no statistically significant differences in the percentage of patients in whom the etiology was demonstrated only in EPS / VB3 or only in the semen (Fischer's exact test, p = 0.1454). Since the results of this study indicate that a large proportion of patients with chronic prostatitis have causitive pathogen that is only proven by semen dijagnostics, not by EPS and / or VB3 (ie. 'four glass test' or 'two glass test'), we conclude that the semen microbiological diagnostics in these patients could constitute a valuable supplement to existing methods (making it a 'five glass test').