Multiple sclerosis (MS) is characterized by a wide interpatient clinical variability and available biomarkers of disease severity still have suboptimal reliability. We aimed to assess immunological and MRI-derived measures of brain tissue damage in patients with different motor impairment degrees, for in vivo investigating the pathogenesis of MS-related disability. Twenty-two benign (B), 26 secondary progressive (SP), and 11 early, nondisabled relapsing-remitting (RR) MS patients and 37 healthy controls (HC) underwent conventional and diffusion tensor brain MRI and, as regards MS patients, immunophenotypic and functional analysis of stimulated peripheral blood mononuclear cells (PBMC). Corticospinal tract (CST) fractional anisotropy and grey matter volume were lower and CST diffusivity was higher in SPMS compared to RRMS and BMS patients. CD14+IL6+ and CD4+IL25+ cell percentages were higher in BMS than in SPMS patients. A multivariable model having EDSS as the dependent variable retained the following independent predictors: grey matter volume, CD14+IL6+ and CD4+IL25+ cell percentages. In patients without motor impairment after long-lasting MS, the grey matter and CST damage degree seem to remain as low as in the earlier disease stages and an immunological pattern suggestive of balanced pro- and anti-inflammatory activity is observed. MRI-derived and immunological measures might be used as complementary biomarkers of MS severity.