Our study evaluated the frequency of developing multiple sclerosis (MS) after acute isolated optic neuritis (ON), the possible association with risk factors (gender, age), and the diagnostic and prognosis role of paraclinical tests. We studied 100 ON patients (mean age 28.9 years: SD 8.9): 85 patients were regularly followed up. Sixty-six patients underwent multimodality evoked potential (EP) test, examination and brain MRI within six months of the onset of ON. Over a mean follow-up of 5.2 years, MS occurred in 28/85 cases. The risk of developing MS after four years was 0.35 at life-table analysis, regardless of gender or age at the onset of ON. Visual EPs in unaffected eyes were abnormal in 25.4%, brainstem auditory EPs in 6.5% somatosensory EPs in 8.1%, upper limb motor EPs in 6.8% of the tested patients; intrathecal IgG synthesis was revealed in 51.7% and MRI lesions in 73.8%. Fifty-one of the patients who underwent paraclinical tests were followed up for more than one year, and MS occurred in 13 cases. All of these presented MRI lesions, nine intrathecal IgG synthesis, and two abnormal extraocular EPs. The risk of developing MS after four years was 0.33 in patients with MRI lesions; the simultaneous presence of intrathecal IgG synthesis increased the risk to 0.46.