Multiple sclerosis (MS) is a chronic demyelinating disease characterised by immune cell infiltration resulting in lesions that preferentially affect periventricular areas of the brain. Despite research efforts to define the role of various immune cells in MS pathogenesis, the focus has been on a few immune cell populations while full-spectrum analysis, encompassing others such as natural killer (NK) cells, has not been performed. Here, we used single-cell mass cytometry (CyTOF) to profile the immune landscape of brain periventricular areas - septum and choroid plexus - and of the circulation from donors with MS, dementia and controls without neurological disease. Using a 37-marker panel, we revealed the infiltration of T cells and antibody-secreting cells in periventricular brain regions and identified a novel NK cell signature specific to MS. CD56bright NK cells were accumulated in the septum of MS donors and displayed an activated and migratory phenotype, similar to that of CD56bright NK cells in the circulation. We validated this signature by multiplex immunohistochemistry and found that the number of NK cells with high expression of granzyme K, typical of the CD56bright subset, was increased in both periventricular lesions and the choroid plexus of donors with MS. Together, our multi-tissue single-cell data shows that CD56bright NK cells accumulate in the periventricular brain regions of MS patients, bringing NK cells back to the spotlight of MS pathology.