Background The yield of next generation sequencing (NGS) added to a Sanger sequencing-based 16S ribosomal RNA (rRNA) gene PCR assay was evaluated in clinical practice for diagnosis of bacterial infection. Methods PCR targeting the V1 to V3 regions of the 16S rRNA gene was performed, with amplified DNA submitted to Sanger sequencing and/or NGS (Illumina MiSeq), or reported as negative, depending on cycle threshold (Ct) value. 2,146 normally sterile tissues or body fluids were tested between August 2020 and March 2021. Clinical sensitivity was assessed in 579 subjects from whom clinical data was available. Results Compared to Sanger sequencing alone (400 positive tests), positivity increased by 87% by adding NGS (347 added positive tests). Clinical sensitivity of the assay incorporating NGS was 53%, higher than culture (42%, p<0.001), with an impact on clinical decision-making in 14% of infected cases. Clinical sensitivity in the subgroup receiving antibiotics at sampling was 41% for culture and 63% for the sequencing assay (p<0.001). Conclusion Adding NGS to Sanger sequencing of the PCR-amplified 16S rRNA gene substantially improved test positivity. In the patient population studied, the assay was more sensitive than culture, and especially so in patients who had received antibiotic therapy.