Bacterial infections pose significant global health challenges, often underestimated due to difficulties in accurate diagnosis, especially when culture-based diagnostics fail. This study assesses the effectiveness of 16S-based metagenomic next generation sequencing (NGS) for identifying pathogens in culture-negative clinical samples across various medical settings. Overall, 48% of samples were collected from orthopedics, 15% from neurosurgery, and 12% in cardiac surgery, among others. The detection rate of monomicrobial infections was 68.6%, and 5.7% for polymicrobial infections. In addition, NGS detected bacteria in all samples from the lungs, head and neck, and eye specimens. (11%, 12/105) was the most frequent microorganism, followed by (10.4%, 11/105), and (9.5%, 10/105). In conclusion, 16S-targeted metagenomic sequencing enhances pathogen detection capabilities, particularly in instances where traditional cultures fail. By the combination of NGS and bacterial cultures, microbiologists might provide a more accurate diagnosis, guiding more effective treatments and potentially reducing healthcare costs associated with empirical treatments.