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<i>Usnea barbata</i> (L.) Weber ex F.H. Wigg (<i>U. barbata</i>) is a medicinal representative of the lichens from the <i>Usnea</i> genus (<i>Parmeliaceae,</i> lichenized <i>Ascomycetes</i>), containing bioactive secondary metabolites. The aim of this study is a comparative analysis between two separated parts of the thallus layers: medulla–cortex (<i>mc</i>UB) and central cord (<i>cc</i>UB) and the whole dried <i>U. barbata</i> thallus (<i>d</i>UB). These three samples were examined regarding color differences. The <i>U. barbata</i> thallus morphology was examined through fluorescent microscopy (FM) and scanning electron microscopy (SEM). The mineral content was measured using inductively coupled plasma mass spectrometry (ICP-MS), and Fourier transform infrared spectroscopy (FT-IR) preliminarily established the differences in the metabolite content. Finally, extracts in different solvents (ethanol and acetone) were obtained from all studied samples, and their total phenolic content (TPC) and free radical scavenging activity (antiradical activity, AA) were evaluated by spectrophotometry. The ICP-MS results showed that from 23 elements analyzed, 18 minerals were quantified in <i>mc</i>UB, 13 in <i>d</i>UB, and only 12 in <i>cc</i>UB. The <i>cc</i>UB fraction recorded the lowest mineral content, color intensity (chroma), luminosity (<i>L</i>*), and TPC value, followed in increasing order by <i>d</i>UB and <i>mc</i>UB. FT-IR spectra displayed different peaks in <i>cc</i>UB and <i>d</i>UB samples compared to <i>mc</i>UB. The <i>mc</i>UB fraction also showed the highest TPC, significantly correlated with AA. However, <i>d</i>UB had the highest antiradical activity, followed by <i>mc</i>UB and <i>cc</i>UB, with noticeable differences in the acetone extract. The final correlation between all variable data obtained indicates that 99.31% of the total variance was associated with all minerals, total phenolics, and color parameters and was also related to the antiradical activity. These obtained results complete our previous studies on autochthonous <i>U. barbata.</i> Moreover, being a source of bioactive metabolites, extracting them from the <i>mc</i>UB fraction could increase this process’s yield and selectivity.