Introduction: The risk of developing depression is increased in patients with autoimmune thyroiditis. Autoimmune Hashimoto thyroiditis is diagnosed using the serum markers anti-thyroid peroxidase (TPO) and anti-thyroglobulin (TG) antibodies. In rare cases, patients with autoimmune thyroiditis can also suffer from the heterogeneous and ill-defined syndrome of Hashimoto encephalopathy. Biomarkers for Hashimoto encephalopathy or for any brain involvement of autoimmune thyroiditis are currently lacking. The aim of the present descriptive study was therefore to determine whether a subgroup of seropositive patients shows intrathecal anti-thyroid antibody synthesis in the cerebrospinal fluid (CSF). Participants and methods: Paired serum and CSF samples from 100 patients with unipolar depression were examined for anti-TPO and anti-TG antibodies using enzyme-linked immunosorbent assays. Antibody-specific indices (ASIs) were calculated for seropositive samples. These ASIs allow the differentiation between the brain-derived fraction of antibodies and antibodies which are passively diffused from the serum. ASIs >1.4 were assessed as positive for brain-derived antibodies. Additionally, for explorative evaluations, a stricter ASI limit of >2 was applied. Results: Anti-TPO antibodies were increased in the serum of 16 patients (16%); increased anti-TPO ASIs (>1.4) were detected in 11 of these patients (69%). Anti-TG antibodies in the serum were detected in three patients (3%), with two of them (67%) showing increased ASIs (>1.4). Overall, the authors found increased anti-thyroid antibodies in 17 of 100 patients (17%), with 13 out of 17 patients showing increased ASIs (76%; range 1.4–4.1). Choosing ASI levels of >2 led to positive findings in six out of 16 patients (38%) with anti-TPO antibodies in their serum but no increase in ASIs in three patients (0%) who were seropositive for anti-TG antibodies. The patients with elevated ASIs (N = 13) were younger than the ASI-negative patients (N = 87; p = 0.009); no differences were noted in the frequency of CSF, electroencephalography, and/or magnetic resonance imaging alterations. Discussion: A subgroup of seropositive patients showed intrathecal synthesis of anti-TPO and, more rarely, of anti-TG antibodies, which might be an indication of central autoimmunity in a subgroup of patients with unipolar depression. The confirmation of elevated ASIs as a biomarker for Hashimoto encephalopathy must await further studies. The relevance of the findings is limited by the study’s retrospective and uncontrolled design.