PURPOSE: Accurate identification of pathogens is critical for clinical microbiology laboratory. This helps in infection prevention and control and antimicrobial stewardship. The study objective was to develop and evaluate the importance of 16S ribosomal RNA sequencing for the identification of fastidious bacteria which are difficult to identify by conventional biochemical methods. This study also assessed the impact of this technology on rationalisation of antimicrobial therapy. MATERIALS AND METHODS: Clinical isolates or samples were chosen for 16S rRNA polymerase chain reaction (PCR) and Sanger sequencing based on the inability to detect or identify pathogens by conventional culture-based methods. This study was done on 148 patients for whom 16S rRNA PCR-Sanger sequencing was performed prospectively on clinical isolates/specimens from August 2015 to June 2017 in a tertiary cancer care hospital in Eastern India. Sanger sequencing was done using Applied Biosystems 3500 Genetic Analyzer. DNA sequence-based identification was done using NCBI BLAST. RESULTS: Forty-five different types (genera) of microorganisms were identified by this 16S rRNA PCR-sequencing method. Amongst the obligate aerobic bacteria identified by the 16S PCR sequencing method (n = 73) 55% were Gram positive and 45% Gram negative. Very few obligate anaerobes, capnophilic and microaerophilic bacteria were identified (n = 2, 1 and 6, respectively). Amongst facultatively anaerobic bacteria (n = 66), 64% were Gram negative and 35% Gram positive. The effect of 16S-based identification on antimicrobial stewardship has been documented. CONCLUSION: The study shows the potential importance of 16S rRNA-based bacterial identification as an important tool in clinical microbiology.