In chronic bacterial prostatitis there are bacteria in the prostate, but there may be no symptoms or milder symptoms than occur with acute prostatitis. The prostate infection is diagnosed by culturing urine as well as prostate fluid (expressed prostatic secretions or EPS) which are obtained by the doctor performing a rectal exam and putting pressure on the prostate. If no fluid is recovered after this prostatic massage, a post massage urine should also contain any prostatic bacteria.

Prostate specific antigen levels may be elevated, although there is no malignancy. Semen analysis is a useful diagnostic tool. Semen cultures are also performed. Antibiotic sensitivity testing is also done to select the appropriate antibiotic. Other useful markers of infection are seminal elastase and seminal cytokines.