Purpose To investigate topographical relationship between amplitude of multifocal visual evoked potentials (mfVEP) and retinal nerve fibre layer (RNFL) thickness following acute optic neuritis (ON). Patients and Methods Fifty patients with a clinical diagnosis of acute unilateral ON between 6 and 36 months prior to the study and 25 age-matched controls underwent mfVEP testing (Accumap V 2.1, ObjectiVision Pty Ltd, Sydney, Australia) and OCT imaging (fast RNFL protocol, Stratus™, software version 3.0, Carl Zeiss Meditec, Inc., Dublin, CA). RNFL thickness and mfVEP amplitude were measured for upper, temporal and lower retinal sectors and corresponding areas of the visual field in affected eyes of ON patients and control eyes. Inter-eye asymmetry coefficients for both RNFL thickness and mfVEP amplitude were calculated for each zone, and corresponding coefficients were correlated between each other. Results There was highly significant reduction of RNFL thickness and mean mfVEP amplitude in all three retinal sectors of the affected eye. Largest reduction of RNFL thickness was noticed in temporal sector and of mfVEP amplitude in corresponding central part of the visual field. RNFL thickness correlated highly with amplitude of the mfVEP derived from corresponding areas of the visual field in all three zones. Conclusions We demonstrated strong topographical associations between structural and functional measures of optic nerve integrity in patients with ON.