Next-generation sequencing-based 16S rRNA gene metagenomic sequencing (16S MG) technology has tremendous potential for improving diagnosis of bacterial infections given its quantitative capability and culture-independent approach. We validated and used a quantitative 16S MG assay to identify and quantify bacterial species in clinical samples from a wide spectrum of infections, including meningitis, septic arthritis, brain abscess, intra-abdominal abscess, soft tissue abscess, and pneumonia. Twenty clinical samples were tested, and 16S MG identified a total of 34 species, compared with 22 species and three descriptive findings identified by culture. 16S MG results matched culture results in 75% (15/20) of the samples but detected at least one more species in five samples, including one culture-negative cerebrospinal fluid sample that was found to contain Streptococcus intermedius. Shotgun metagenomic sequencing verified the presence of all additional species. The 16S MG assay is highly sensitive, with a limit of detection of 10 to 100 colony-forming units/mL. Other performance characteristics, including linearity, precision, and specificity, all met the requirements for a clinical test. This assay showed the advantages of accurate identification and quantification of bacteria in culture-negative and polymicrobial infections for which conventional microbiology methods are limited. It also showed promises to serve unmet clinical needs for solving difficult infectious diseases cases.