Background: Progressive Multifocal Leukoencephalopathy (PML) is an opportunistic infection caused by John Cunningham virus (JCV) reactivation, potentially associated with natalizumab (NTZ) treatment for Multiple Sclerosis (MS). The anti-JCV antibodies titre (JCV index) increases during NTZ treatment; however, the effects of other disease-modifying therapies (DMTs) on the JCV index have not been fully explored. Objective: The aim of the study was to evaluate changes in the JCV index during treatment with several DMTs. Methods: This longitudinal study evaluated the JCV index before starting DMT (T0) and during treatment with DMT (T1). Results: A total of 260 participants (65.4% females, mean age 43 ± 11.3) were enrolled: 68 (26.2%) treated with fingolimod (FTY), 65 (25%) rituximab or ocrelizumab (RTX/OCR), 37 (14.2%) dimethyl-fumarate (DMF), 29 (11.2%) cladribine (CLD), 23 (8.8%) teriflunomide (TFM), 20 (7.7%) interferon or glatiramer acetate (IFN/GA), and 18 (6.9%) alemtuzumab (ALM). At T1, the percentage of patients with JCV index <0.90 was found to be significantly increased in the ALM group (16.7% versus 66.7%, p = 0.05), while the percentage of patients with JCV index >1.51 was found to be significantly reduced in the RTX/OCR group (51.6% versus 37.5%, p = 0.04). In the FTY group, a significant reduction in the percentage of patients with JCV index <0.90 was also found (23.5% versus 1.4%, p = 0.0006). The mean JCV index was reduced in the RTX/OCR and ALM groups, while a significant increase was observed in the FTY group. Conclusion: DMTs with a T and/or B depleting mechanism of action induced a significant reduction in the JCV index. These results may suggest new possible sequencing strategies potentially maximizing disease control while reducing the PML risk.