Background Anti-streptavidin antibodies are causal determinants of analytical interference during Thyroid function tests, and numerous reports have detailed such interference, with anti-streptavidin antibodies attracting attention. Case presentation We conducted a straightforward investigation of interference due to anti-streptavidin antibodies, with a case of a 60-year-old Japanese man who consulted our department for inconsistencies between his clinical course and Thyroid function tests. Experiments were conducted using Cobas8000 e602, which employs assay procedures with pre-wash to evaluate FT4 and FT3 levels. Conclusions To our knowledge, this is the first published report to clearly investigate such interferences using a combination of polyethylene glycol precipitation, heterophilic blocking tube precipitation, streptavidin-coated magnetic particle precipitation, and different instruments with or without pre-wash. Clinicians should consider that interferences caused by anti-streptavidin antibodies could lead to a misdiagnosis of thyrotoxicosis. Moreover, discussions between laboratory specialists, clinicians, and manufacturers are required to identify interferences and avoid unnecessary examinations and inappropriate treatment.