Abstract Objective Diagnosing septic arthritis can be challenging and frequently involves clinical assessment, laboratory investigations and synovial fluid analysis. We sought to determine the utility of synovial aspiration and intra-operative synovial fluid and tissue culture for the accurate diagnosis of septic arthritis. Methods We carried out a retrospective review of the records of patients referred to a tertiary orthopaedic unit with possible septic arthritis between 2015 and 2019 inclusive, including clinical and laboratory data for this cohort study. Performance characteristics were determined for synovial aspiration, intra-operative synovial fluid and tissue culture in diagnosing expert review-determined true septic arthritis. Concordance between discharge diagnosis, antibiotic prescribing and true septic arthritis was determined. Results Of 268 patients identified with suspected septic arthritis, 143 underwent both synovial fluid aspiration and intra-operative synovial fluid and tissue biopsy culture. True septic arthritis was not differentiated significantly by laboratory parameters including serum white cell count (WCC), CRP or synovial WCC. Considering only patients with negative pre-operative synovial aspirate cultures, intra-operative samples led to diagnosis of true septic arthritis in 6 of 63 patients [number needed to treat (NNT) 10.5]. For all patients sampled in theatre, positive synovial tissue biopsy was the only evidence of true septic arthritis in six (NNT 23.9). Despite insufficient microbiological evidence, 27 of the 59 patients who did not have septic arthritis received a discharge diagnosis of septic arthritis, 26 of whom were discharged with antibiotics. Conclusion Intra-operative sample collection, particularly tissue biopsy, increases the likelihood of a true septic arthritis diagnosis. Such measures might help to reduce diagnostic ambiguity in clinical practice and might therefore reduce overtreatment.