(1) To evaluate the diagnostic testing performance of the synovial white blood cell (WBC) count, polymorphonuclear cell percentage, and synovial glucose, synovial protein, synovial lactate dehydrogenase, and synovial C-reactive protein levels as diagnostic markers for the diagnosis of septic arthritis after anterior cruciate ligament (ACL) reconstruction; (2) to define the ideal thresholds of the aforementioned tests, leading to the optimal sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy; and (3) to define the sensitivity of synovial fluid culture and synovial tissue sample culture, as well as determine whether previous antibiotic treatment may affect the accuracy of these tests. We performed a retrospective analysis of all patients readmitted from January 2009 to September 2019 with signs suggestive of septic arthritis and undergoing a knee aspiration for synovial fluid analysis and culture. The receiver operating characteristic curve and the associated area under the curve were constructed for the aforementioned synovial markers. Sensitivity, specificity, PPV, NPV, and accuracy were calculated for the obtained optimal values. Sensitivity was also calculated for synovial fluid culture and synovial tissue sample culture, and the influence of previous antibiotic treatments on culture sensitivity was evaluated. Among 3,408 cases of ACL reconstruction, after the exclusion of 13 patients not meeting the inclusion criteria, 24 infected and 14 uninfected patients were reviewed and included in the analysis. The diagnosis was confirmed by the presence of 2 positive culture findings with the same isolated microorganism or at least 3 of the 4 following criteria: elevated serum C-reactive protein level and erythrocyte sedimentation rate, positive results of histologic analysis of synovial tissue, macroscopic evidence of purulence, and 1 positive culture finding. The receiver operating characteristic curve analysis showed that the most reliable marker for the diagnosis of septic arthritis after ACL reconstruction was the synovial WBC count (area under the curve, 0.89). A cutoff value of 28,100 cells/mL presented the highest accuracy (0.85), highest PPV (0.94), and highest NPV (0.76); moreover, with the threshold set at 40,000 cells/mL, postoperative infection could be diagnosed with 100% specificity. The sensitivity of synovial fluid culture was significantly lower than the sensitivity of synovial tissue sample culture (0.63 vs 0.96, P= .0045); moreover, the sensitivity further decreased if patients took antibiotics before aspiration (0.44 vs 0.73), although this decrease was not statistically significant. The synovial WBC count is the most reliable test for the diagnosis of septic arthritis after ACL reconstruction. Although the sensitivity of synovial fluid culture is affected by previous antibiotic treatment, the synovial WBC count is not influenced and proves to be useful in the diagnosis of this uncommon complication. Level II, diagnostic study.