Local synthesis of antibodies and presence of oligoclonal bands in the cerebrospinal fluid (CSF) are hallmarks of multiple sclerosis (MS). We investigated the frequency of antibodies against mycobacterial and relevant human epitopes in the CSF of patients with MS or neuromyelitis optica spectrum disorder (NMOSD) and whether these antibodies differed from those present in the serum. Matched serum and CSF samples from 46 patients with MS, 42 patients with NMOSD, and 29 age-matched and sex-matched control subjects were screened retrospectively for the presence of antibodies against Mycobacterium avium subsp. paratuberculosis (MAP) pentapeptide (MAP_5p), MAP_2694295⁻303, and myelin basic protein (MBP)85⁻98 peptides by using indirect ELISA. Serum levels of anti-MAP_5p and anti-MAP_2694295⁻303 antibodies were highly prevalent in patients with MS when compared to patients with NMOSD and controls. Several patients with MS had detectable anti-MAP_5p and anti-MAP_2694295⁻303 antibodies in the CSF. Furthermore, a group of patients with MS showed intrathecally restricted production of antibodies against these peptides. Women appeared to mount a stronger humoral response to mycobacterial peptides than men. No significant difference in the frequency of anti-MBP85⁻98 antibodies was found between patients with MS and those with NMOSD. These data highlight the zoonotic potential of MAP, which suggests its involvement in MS etiopathogenesis.