Introduction: Abnormal liver function tests are frequently seen in patients with multiple sclerosis (MS) and their origin at times is attributed to the possible co-occurrence or the de novo induction of autoimmune liver diseases (AILD), namely autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC), but comprehensive analysis of AILD-related autoantibody has not been carried out. Aim: To assess the presence of AILD-related autoantibodies in a well-defined cohort of MS patients, and to assess their clinical significance. Materials and methods: 133 MS (93 female) patients (102 RRMS, 27 SPMS, and 5 PPMS), mean age 42.7 ± 11.9 SD years, mean duration of disease 11.2 ± 7.2 years were studied. 150 age and sex-matched healthy individuals were tested as normal controls (NCs).Autoantibody testing was performed by indirect immunofluorescence (IF) using triple tissue and HEp-2, a multiparametric line immunoassay detecting anti-LKM1(anti-CYP2D6), anti-LC1(anti-FTCD), soluble liver antigen/liver-pancreas(anti-SLA/LP), AMA-M2, and AMA-MIT3 (BPO), PBC-specific ANA (anti-gp210, anti-sp100 and anti-PML), and ELISA for anti-F-actin SMA and anti-dsDNA antibodies. Results: Reactivity to at least one autoantibody was more frequent in MS patients compared to NCs (30/133, 22.6% vs 12/150, 8%) NCs (p = 0.00058). SMAs by IIF were more frequent in MS patients (18/133, 13.53%) compared to NCs (6/150, 4%, p = 0.002%). The AIH-1 related anti-F-actin SMA by ELISA were present in 21 (15.8%), at relatively low titres (all but three of the SMA-VG pattern by IF); anti-dsDNA in 3 (2.3%), and anti-SLA/LP in none; AIH-2 anti-LKM1 autoantibodies in 1 (0.8%, negative by IF), and anti-LC1 in none; PBC-specific AMA-M2 in 2 (1.5%, both negative for AMA-MIT3 and AMA by IF) and PBC-specific ANA anti-PML in 6 (4.5%), anti-sp100 in 1 (0.8%) and anti-gp210 in 1 (0.8%). Amongst the 30 MS patients with at least one autoantibody positivity, only 4 (3%) had overt AILD (2 AIH-1 and 2 PBC). Autoantibody positivity did not differ between naïve MS patients and patients under treatment.