Background: Immune checkpoint inhibitors (ICIs) are associated with several endocrine side effects. In particular, the use of programmed cell death protein-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) inhibitors is related to a higher incidence of thyroid dysfunction. Patient Findings: An 85 years-old patient, diagnosed with a metastatic melanoma treated with nivolumab, presented to our hospital with severe ICI-related thyrotoxicosis. Diagnosis was complicated by a biochemical interference on thyroid hormones assay, probably induced by nivolumab. Summary: Baseline laboratory examination conducted before onset of anticancer therapy showed normal thyroid function test (TFTs). A few days after receiving the second nivolumab administration, the patient developed a severe thyrotoxicosis. According to destructive thyroiditis, in a short period thyrotropin (TSH) levels normalized and rapidly increased, but free thyroxine (fT4) levels were inappropriately elevated and did not decrease as expected. The sample was processed by using a Siemens Centaur® immunoassay. We reanalyzed the same sample at another laboratory and with a different immunoassay method (Roche Elecsys®). The results obtained from this assay confirmed severe hypothyroidism with appropriately low fT4 levels. We suspected a possible nivolumab-associated interference on the fT4 assay. Therefore, we subjected the same sample to a polyethylene glycol (PEG) 6000 precipitation, a simple method for the removal of macromolecules, before assaying for fT4 levels. Evaluation of the post-PEG-precipitation sample (Siemens Centaur immunoassay) revealed appropriately low fT4 levels. The patient was started on levothyroxine (LT4) therapy, with monthly TFT monitoring using the Roche immunoassay. Approximately 9 months after starting nivolumab therapy, the patient was advised treatment cessation. A month later, the TFTs were retested on a Siemens Centaur immunoassay, and appropriate fT4 levels were observed in accordance with normal TSH levels on adequate LT4 replacement therapy. Conclusions: We report a possible novel nivolumab-induced biochemical interference on assays of fT4 levels. The hypothesis of a biochemical drug-induced interference is further supported by the disappearance of the interference after the withdrawal of nivolumab. Further studies are needed to prove the biochemical mechanisms of this interference.