Objective: To elucidate cross-sectional patterns and longitudinal changes of oral and stool microbiota in patients with multiple sclerosis (MS) and the effect of B-cell depletion.Methods: We conducted an observational, longitudinal clinical cohort study analyzing four timepoints (days 0 and 14, weeks 24 and 52) over 12 months in 37 MS patients, of whom 22 initiated B-cell depletion therapy with ocrelizumab and 15 age- and gender-matched untreated MS patients. For microbiota analysis of the oral cavity and the gut, all provided stool and oral swab samples underwent 16S rDNA sequencing and subsequent bioinformatic analyses. Clinical outcome and MRI results were registered. Results: Compared to healthy controls, MS patients showed decreased alpha-diversity, significantly increased detection of Bacteroidetes and lower abundance of butyrate-producing members of the Firmicutes in their stool. Oral microbiota-patterns also had a reduced alpha-diversity yet showed unique and differential microbiota changes compared to stool such as increased levels of Proteobacteria and decreased abundance of Actinobacteria. Remarkably, following initiation of B-cell depletion, we observed increased alpha-diversity in the gut and the oral cavity as well as a long-term sustained reduction of pro-inflammatory Gram-negative bacteria (e.g., Escherichia/Shigella).Conclusion: MS patients have altered stool and oral microbiota diversity patterns compared to healthy controls, which are most pronounced in patients with higher disease activity and disability. Therapeutic B-cell depletion is associated with persisting regression of these changes. Whether these microbial changes are unspecific side-effects of B-cell depletion or indirectly modulate multiple sclerosis disease activity and progression is currently unknown and necessitates further investigations.