Background: Mycobacterium avium subspecies paratuberculosis (MAP) is an infectious factor recently found in association with multiple sclerosis (MS) in Sardinia. Objectives: The objectives of this study were to confirm this association and evaluate its role in clinical features. Methods: A total of 436 patients and 264 healthy controls (HCs) were included. We examined the blood of each individual for MAPDNA and MAP2694 antibodies using IS900-specific PCR and ELISA, respectively. Differences in MAP presence between the MS group and HCs were evaluated. In MS patients, we considered: gender, age, age at onset, duration of disease, course, EDSS, therapy, relapse/steroids at study time, and oligoclonal bands (OBs). Results: MAPDNA and MAP2694 antibodies were detected in 68 MS and six HCs ( p = 1.14 × 10−11), and 123 MS and 10 HCs ( p = 2.59 × 10−23), respectively. OBs were found with reduced frequency in MAP-positive patients (OR = 0.52; p = 0.02). MAP2694 antibodies were detected more in patients receiving MS treatments (OR = 2.26; p = 0.01), and MAPDNA in subjects on steroids (OR = 2.65; p = 0.02). Conclusion: Our study confirmed the association of MAP and MS in Sardinia. The low OB frequency in MAP patients suggests a peripheral role as a trigger in autoimmunity. MAP positivity might be influenced by steroids and MS therapy. Studies in other populations are needed to confirm the role of MAP in MS.