Background Studying axonal loss in the retina is a promising biomarker for multiple sclerosis (MS). Our aim was to compare optical coherence tomography (OCT) and Heidelberg retinal tomography (HRT) techniques to measure the thickness of the retinal nerve fiber layer (RNFL) in patients with MS, and to explore the relationship between changes in the RNFL thickness with physical and cognitive disability. We studied 52 patients with MS and 18 proportionally matched controls by performing neurological examination, neuropsychological evaluation using the Brief Repetitive Battery-Neuropsychology and RNFL thickness measurement using OCT and HRT. Results We found that both OCT and HRT could define a reduction in the thickness of the RNFL in patients with MS compared with controls, although both measurements were weakly correlated, suggesting that they might measure different aspects of the tissue changes in MS. The degree of RNFL atrophy was correlated with cognitive disability, mainly with the symbol digit modality test ( r = 0.754, P < 0.001). Moreover, temporal quadrant RNFL atrophy measured with OCT was associated with physical disability. Conclusion In summary, both OCT and HRT are able to detect thinning of the RNFL, but OCT seems to be the most sensitive technique to identify changes associated with MS evolution.