Simple Summary: Natural killer cells and cytotoxic CD8+ T cells play a complementary role in controlling cytomegalovirus, a ubiquitous herpesvirus that establishes a lifelong latent infection in the host. Due to its ability to modulate the immune response, cytomegalovirus can impact the course of multiple sclerosis, an immune-mediated disease of the central nervous system. However, whether this herpesvirus may play a beneficial or a detrimental role in multiple sclerosis is currently uncertain. The cytomegalovirus seropositivity status increases both the expansion of highly differentiated CD8+ T cells and the number of natural killer cells expressing the NKG2C receptor. Given that NKG2C is an activating receptor that enhances natural killer cell-mediated cytotoxicity and recruitment of inflammatory cells, it is reasonable to investigate the involvement of this receptor in immune-mediated diseases. In the present study, we explored the magnitude of cytomegalovirus-induced immune changes in multiple sclerosis in order to increase the knowledge of the relationship between viruses and immune-mediated diseases. Multiple sclerosis (MS) is a debilitating neurological disease that has been classified as an immune-mediated attack on myelin, the protective sheath of nerves. Some aspects of its pathogenesis are still unclear; nevertheless, it is generally established that viral infections influence the course of the disease. Cytomegalovirus (CMV) is a major pathogen involved in alterations of the immune system, including the expansion of highly differentiated cytotoxic CD8+ T cells and the accumulation of adaptive natural killer (NK) cells expressing high levels of the NKG2C receptor. In this study, we evaluated the impact of latent CMV infection on MS patients through the characterization of peripheral NK cells, CD8+ T cells, and NKT-like cells using flow cytometry. We evaluated the associations between immune cell profiles and clinical features such as MS duration and MS progression, evaluated using the Expanded Disability Status Scale (EDSS). We showed that NK cells, CD8+ T cells, and NKT-like cells had an altered phenotype in CMV-infected MS patients and displayed high levels of the NKG2C receptor. Moreover, in MS patients, increased NKG2C expression levels were found to be associated with higher EDSS scores. Overall, these results support the hypothesis that CMV infection imprints the immune system by modifying the phenotype and receptor repertoire of NK and CD8+ T cells, suggesting a detrimental role of CMV on MS progression. [ABSTRACT FROM AUTHOR]