Thyroid Benign Nodules (TBN) are the most common lesions of this endocrine gland. The etiology of TBN is not clear. The aim of this exploratory study was to examine differences in the content of bromine (Br), cooper (Cu), iron (Fe), iodine (I), rubidium (Rb), strontium (Sr), and zinc (Zn), as well as I/Br, I/Cu, I/Fe, I/Rb, I/Sr, and I/Zn content ratios in tissues of normal thyroid and TBN. Thyroid tissue levels of seven Trace Elements (TEs) were prospectively evaluated in 105 apparently healthy persons and in 79 patients with TBN. Measurements were performed using 109Cd and 241Am radionuclide-induced energy-dispersive X-ray fluorescent analysis. Tissue samples were divided into two portions. One was used for morphological study while the other was intended for TEs analysis. It was observed that in TBN the mass fraction of Br, Cu, and Fe were higher, whereas mass fractions of I as well as I/Br, I/Cu, I/Fe, and I/Zn mass fraction ratios were lower than in normal tissues of the thyroid. These changes can potentially be used as TBN markers. Furthermore, it was found that the levels of Br, Cu, Fe, Rb, Sr, and Zn contents in the normal and affected thyroid gland were interconnected and depend on the content of I in thyroid tissue. Because I plays a decisive role in the function of the thyroid gland, the data obtained allow us to conclude that, along with I, at least such TEs as Br, Cu, Fe, Rb, and Zn, if not directly, then indirectly, are involved in the process of thyroid hormone synthesis.