Different types of infection are implicated in the pathogenesis of autoimmune thyroid diseases (AITD) through molecular mimicry or other mechanisms, but their role is disputed. Human studies support direct or indirect evidence of involvement of some viral and bacterial agents, but reports have provided conflicting and inconclusive results. Using a new automated multiplex array platform for the detection of antibodies, we determined seroreactivity against Toxoplasma gondii, Treponema pallidum, rubella virus, cytomegalovirus, and Epstein-Barr virus in a large group of Italian AITD patients and healthy controls. Only IgG concentrations against T. gondii were significantly higher in AITD patients than in controls, suggesting that these protozoa may be involved in the initiation of both Hashimoto's thyroiditis and Graves' disease.