Nested PCR method was used in the present study for detection of pathogenic bacteria in clinical body fluids. This method permitted a reliable amplification of 16S rRNA of common bacterial pathogens in body fluids with 1 CFU mL-1 sensitivity threshold in detection. A total of 36 clinical fluid specimens (9 peritoneal, 9 synovial, 9 pleural and 9 cerebrospinal fluids) were analyzed. Bacterial pathogens in 26 specimens were successfully detected by culture-based method and by nested PCR method. Whereas, DNA extracts from seven fluid specimens which showed no bacterial growth on conventional media were positively amplified by nested PCR. Thus, nested PCR method proved to be more rapid and more sensitive than culturing method in detection of bacterial pathogens, including fastidious and low levels of bacteria, in clinical body fluids. In conclusion, nested PCR method is worth considering in detection protocols of bacterial pathogens in clinical body fluids.