Aims: This study was aimed to directly diagnose bacterial pathogens from clinical specimens by amplifying and sequencing their 16S rRNA gene compared with traditional methods. Methodology and results: A total of 100 specimens, including blood (50 samples) and cerebrospinal fluid CSF (50 samples) were collected from patients hospitalized in Mosul city hospitals for the period from December 2021 to April 2022. Traditional methods based on cultural characteristics and simple biochemical tests were used to detect pathogens from these samples, as followed by hospital laboratories. Bacterial genomic DNA (deoxyribonucleic acid) was extracted directly from the samples and PCR (polymerase chain reaction) was conducted to amplify their 16S rRNA gene using 16S universal primers. PCR products from samples that showed positive results were purified and sent for sequencing. Sequences were compared to counterpart sequences submitted in NCBI to identify the bacterial pathogen. Our results showed that traditional methods for bacterial isolation and identification from CSF detected 5 positive bacterial cultures out of 50 samples tested (10%), while the remaining cultures (90%) did not show any bacterial growth after 7 days of incubation. The 45 negative samples were subjected to molecular identification and it was found that 28/45 (62.2%) successfully amplified 16S rRNA bands indicating the presence of bacterial genomic DNA. Sequencing results showed that the highest prevalent genera in CSF samples were Bacteroides (41.1%), followed by Lactobacillus (17.6%), Pseudomonas (11.7%), Janibacter (11.7%), Achromobacter (11.7%) and Planococcus (5.8%). Traditional methods for bacterial isolation and identification from blood were capable of isolating 2 pathogens from 50 samples tested (4%). When testing the 48 negative samples using molecular methods, 25/48 (52%) successfully amplified 16S rRNA bands. The 16S rRNA sequences belonged to 9 different genera, the most dominant was Pseudomonas (16.6%) and the least dominant was Bacteroides (5.5%). Conclusion, significance and impact of study: Our results clearly show the power of molecular diagnosis compared to traditional methods used at hospitals and show the danger of misdiagnosis. The study showed that the molecular approach is much more efficient than the conventional methods in identifying pathogens as it identified new pathogens that were not previously detected.