Optic neuritis (ON) is a common manifestation of multiple sclerosis (MS) and myelin-oligodendrocyte-glycoprotein IgG-associated disease (MOGAD). This study evaluated the applicability of optical coherence tomography (OCT) for differentiating between both diseases in two independent cohorts. One hundred sixty two patients from seven sites underwent standard OCT and high-contrast visual acuity (HCVA) testing at least 6months after first ON. Of these, 100 patients (32 MOGAD, 68 MS) comprised the primary investigational cohort, while 62 patients (31 MOGAD, 31 MS) formed a validation cohort. A composite score distinguishing between MOGAD and MS was developed using multivariate logistic regression. Bilateral simultaneous ON occurred more frequently in MOGAD compared to MS (46.9 vs. 11.8%, p < 0.001). OCT revealed more peripapillary retinal nerve fiber layer (pRNFL) atrophy in all segments in MOGADcompared to predominantly temporal pRNFL atrophy in MS (p < 0.001). HCVA was better preserved in MS (p = 0.007). pRNFL thickness in all except for temporal segments was suitable for differentiating MOGAD and MS. Simultaneous bilateral ON and critical atrophy in nasal (< 58.5µm) and temporal superior (< 105.5µm) segments were included into the composite score as three independent predictors for MOGAD. The composite score distinguished MOGAD from MS with 75% sensitivity and 90% specificity in the investigational cohort, and 68% sensitivity and 87% specificity in the validation cohort. Following a single ON-episode, MOGAD exhibits more pronounced global pRNFL atrophy and lower visual acuity after ON compared to MS. The introduced OCT-based composite score enabled differentiation between the two entities across both cohorts.