IMPORTANCE Diagnostic error is an important source of medical error. Overdiagnosis of optic neuritis may prompt unnecessary and costly diagnostic tests, procedures, and treatments. OBJECTIVE To assess the incidence of and characterize factors contributing to overdiagnosis of acute optic neuritis. DESIGN, SETTING, AND PARTICIPANTS In this retrospective clinic-based cross-sectional study of new patient encounters, 122 patients referred for acute optic neuritis at a university-based Midwestern neuro-ophthalmology clinic between January 2014 and October 2016 were studied. Data were analyzed from September 2016 to July 2017. INTERVENTIONS Definite diagnosis was determined by neuro-ophthalmologists. For patients with alterative diagnoses, the Diagnosis Error Evaluation and Research taxonomy tool was applied to categorize the type of diagnostic error. MAIN OUTCOMES AND MEASURES The primary outcomewas the primary type of diagnostic error in patients erroneously diagnosed as having optic neuritis. Secondary outcomes included final diagnosis and interventions undergone prior to referral. RESULTS A total of 122 patients were referred with acute optic neuritis during the study period; 88 (72.1%) were female, and the mean (SD) age was 42.6 (14.0) years. Of these, 49 patients (40.2%; 95%CI, 31.4-49.4) were confirmed to have optic neuritis, and 73 (59.8%; 95%CI, 50.6-68.6) had an alternative diagnosis. The most common alternative diagnoses were headache and eye pain, functional visual loss, and other optic neuropathies, particularly nonarteritic anterior ischemic optic neuropathy. The most common diagnostic error was eliciting or interpreting critical elements of history, which occurred in 24 of 73 patients (33%) with alternative diagnoses. Other common errors included errors weighing or considering alternative diagnoses (23 patients [32%]), errors weighing or interpreting physical examination findings (15 patients [21%]), and misinterpreting diagnostic test results (11 patients [15%]). In patients with alterative diagnoses, 12 (16%) had normal magnetic resonance imaging findings preceding the referral, 12 (16%) had received a lumbar puncture, and 8 (11%) had received unnecessary treatment with intravenous steroids. CONCLUSIONS AND RELEVANCE These data suggest that nearly 60%(95%CI, 50.6-68.6) of patients referred for optic neuritis have an alternative diagnosis, with the most common errors being overreliance on a single item of history and failure to consider alternative diagnoses. Understanding pitfalls leading to overdiagnosis of optic neuritis may improve clinicians' diagnostic process.