Background: Septic arthritis is a debilitating condition with prolonged treatment and adverse outcomes. A gram stain is often performed from the joint aspirate sample, followed by a definitive culture. In our study, we assessed the accuracy of gram staining for suspected septic arthritis and explored factors associated with positive culture growth and false negatives in the gram stain. Methods: We retrospectively reviewed joint aspirates performed from 2015-2021 at a major trauma centre. Aspirates not cultured for septic arthritis were excluded. Data collected included aspirate site, gram stain and culture result delay, patient demographics, orthopaedic/rheumatological history, and comorbidities. Outcomes measured were gram stain sensitivity and specificity. Factors influencing positive cultures and false negative gram stain results were analysed using logistic regression. Results: Of 408 joint aspirates meeting the criteria, 37 did not undergo initial gram staining. Gram stain sensitivity was 30.4%, specificity was 97.6%. The delay from aspirate to definitive gram stain and culture results was 1.1 and 5.4 days, respectively Logistic regression identified that prosthetic joint( p = 0.007), past joint infections( p = 0.006), arthritis( p < 0.001), hypertension( p = 0.007), diabetes( p = 0.019) were positively associated with positive cultures. Past joint infections( p = 0.004) were positively associated with false negative gram stain results. Patients on antibiotics during the aspirate had a higher risk of false negative gram stain results (OR = 5.538, 95%CI, 2.802–10.948; p < 0.001). Conclusions: In conclusion, the initial gram stain has limited sensitivity and caution should be exercised when interpreting negative results. Vigilance is crucial when the highlighted comorbidities or antibiotic use are present, to assess patients with potential joint infections.