Septic arthritis (SA) is a rheumatologic emergency associated with significant morbidity and mortality. Delayed or inadequate treatment of SA can lead to irreversible joint destruction and disability. Current methods of diagnosing SA rely on synovial fluid analysis and culture which are known to be imprecise and time-consuming. We report a novel adaptation of a probe-based real-time PCR assay targeting the 16S rRNA gene for early and accurate diagnosis of bacterial SA. The assay algorithm consists of initial broad-range eubacterial detection, followed by Gram typing and species characterization of the pathogen. The platform demonstrated a high analytical sensitivity with a limit of detection of 101 CFU/ml with a panel of SA-related organisms. Gram typing and pathogen-specific probes correctly identified their respective targets in a mock test panel of 36 common clinically relevant pathogens. One hundred twenty-one clinical synovial fluid samples from patients presenting with suspected acute SA were tested. The sensitivity and specificity of the assay were 95% and 97%, respectively, versus synovial fluid culture results. Gram-typing probes correctly identified 100% of eubacterial positive samples as to gram-positive or gram-negative status, and pathogen-specific probes correctly identified the etiologic agent in 16/20 eubacterial positive samples. The total assay time from sample collection to result is 3 h. We have demonstrated that a real-time broad-based PCR assay has high analytical and clinical performance with an improved time to detection versus culture for SA. This assay may be a useful diagnostic adjunct for clinicians, particularly those practicing in the acute care setting where rapid pathogen detection and identification would assist in disposition and treatment decisions.