CONTEXT: Rapid detection of pathogens in infected body fluids will help in establishing an aetiological diagnosis, thereby facilitating specific therapy. Molecular methods have an advantage over conventional bacteriological techniques in particularly identifying slow-growing, fastidious or non-cultivable organisms. AIM: The aim of this study is to evaluate the use of 16S rRNA polymerase chain reaction (PCR) for detecting bacterial pathogens in body fluids and compare with conventional culture methods. SETTINGS AND DESIGN: This study was done at the Clinical Laboratory of the Department of Microbiology. This was a cross-sectional study design. MATERIALS AND METHODS: A total of 100 consecutive samples which included synovial fluid, cerebrospinal fluid, ascitic fluid and pleural fluid received in the laboratory during the study period were subjected to PCR for 16S rRNA using specific primers and conventional culture by standard protocol. Samples which were positive for 16S rRNA were sequenced to identify the organism. Results of sequenced products were compared in terms of number of organisms, with culture isolates. RESULTS: The detection rate of 16S rRNA PCR was at 13% as compared to culture at 3% (P = 0.0009). The diagnostic sensitivity and specificity of the PCR were 100% and 89.7%, respectively. The concordance of PCR and culture for both identical positive and negative samples was 90%. CONCLUSIONS: The 16S rRNA PCR proved to be rapid method for detection of bacterial pathogens in body fluids. It may be a valuable tool in the diagnostic armamentarium for differentiating bacterial infection from others and starting empiric treatment.