Background: Long-term outcome in multiple sclerosis (MS) depends on early treatment. We investigated markers in cerebrospinal fluid (CSF), which may predict conversion to MS in acute optic neuritis (ON) patients. Methods: Forty patients with acute ON were recruited in a population-based prospective cohort with median 29 months (range 19-41) follow-up. CSF samples were taken prior to treatment within 38 days of onset (median 14 days; range 2-38). Prospectively, 16/40 patients were diagnosed with MS (MS-ON) and 24 patients continued to manifest isolated ON (ION). Levels of cytokines and neurofilament light chain (NF-L) were measured and compared to healthy controls (HC). Significance levels were corrected for multiple comparisons (q). Findings: CSF TNF-α, IL-10 and CXCL13 levels were increased in MS-ON compared to ION patients (q=0·021, 0·004, and 0·0006, respectively). MS-ON patients had increased CSF pleocytosis, IgG indices and oligoclonal bands (OCB) compared to ION (q=0·0007, q=0·0058 and q=0·0021, respectively). CSF levels of IL-10, TNF-a, IL-17A and CXCL13 in MS-ON patients correlated with leukocyte counts (r>0·69 and p 0·55, p<0·037). CSF NF-L levels were increased in ON patients compared to HC (q=0·0077). In MS-ON, an increase in NF-L levels were observed at 7 to 14 days after disease onset (r=0·73, p<0·0065). Receiver operating characteristic (ROC) curves for two multivariable prediction models were generated, with IL-10, CXCL13, and NF-L in one (candidate) and IgG index, OCB, and leukocytes in another (routine). Area under the curve was 0·89 [95% C.I.: 0·77-1] and 0·86 [0·74-0·98], respectively. Predictions of the risk of MS were illustrated by two nomograms. Interpretation: CSF TNF-α, IL-10, CXCL13 and NF-L levels were associated with development of MS, suggesting that the inflammatory and neurodegenerative processes occurred early in acute ON. The nomogram predictions may be useful in clinical diagnostics for MS. Funding: Region of Southern Denmark, Danish MS Society. Declaration of Interest: Mr. Olesen has nothing to disclose; Dr. Soelberg has nothing to disclose; Dr. Debrabant has nothing to disclose; Dr. Nilsson has nothing to disclose; Dr. Grauslund has nothing to disclose; Dr. Brandslund has nothing to disclose; Dr. Madsen has nothing to disclose; Dr. Paul Friedemann reports non-financial support from Novartis, non-financial support and other from Bayer, non-financial support and other from Sanofi Genzyme, non-financial support and other from Merck, non-financial support and other from Roche, outside the submitted work; Terry Smith is scientific advisor to Guthy-Jackson Charitable Foundation; Sven Jarius has nothing to disclose; Dr. Asgari has nothing to disclose. Ethical Approval: The research was carried out in compliance with Danish ethical regulations for clinical research and was approved by The Regional Health Research Ethics Committee for the Region of Southern Denmark (ref. no. S-20130137). All patients provided written informed consent.