The prevalence of autoantibodies in multiple sclerosis (MS) patients and their clinical associations differ between various studies. This study investigated antiphospholipid and antinuclear antibodies in 85 patients with multiple sclerosis (MS) and clinically isolated syndrome (CIS) with regard to their association with demographic features, MS specific clinical features and symptoms of connective tissue diseases. Autoantibodies tested included antinuclear antibodies (ANA) with their specificities and anticardiolipin (aCL) and anti-beta-2-glycoprotein I (anti-β2GPI) antibodies. Antinuclear antibodies were more prevalent in MS patients than in controls (63.5% vs. 3.3%; p < 0.01) and in 19% of patients specific antinuclear antibodies were detected. Anti-β2GPI IgM antibodies were more frequent in MS patients than in the control group (20% vs. 3.3%; p < 0.05). The frequency of anticardiolipin antibodies did not differ between MS patients and controls. MS patients seropositive for ANA and extractable nuclear antigens (ENA) had significantly shorter disease duration than seronegative patients (p < 0.05) and a lower disability score (Expanded Disability Status Score; EDSS) (p < 0.05). Anti-β2GPI antibodies were more frequent in patients with secondary progressive MS (SP-MS) and specific ANA antibodies were more frequent in patients with clinically isolated syndrome (CIS) (p < 0.05). The presence of autoantibodies was not associated with the predominant site of neurological involvement or the clinical features of connective tissue diseases.