<p>Melanoma is a highly aggressive skin cancer with significant metastatic potential. Recent studies highlight the pivotal role of fibroblasts within the melanoma microenvironment. Fibroblasts become activated in response to chronic lesions, such as melanoma, expressing molecules that sustain tumor growth and progression. The symbiotic interaction between fibroblast and tumor cells is complex and not fully elucidated. Despite its importance, few studies have directly compared the biophysical properties of fibroblasts and melanoma cells, particularly in the pulmonary context and in relation to tumor progression. Here, we combined Raman spectroscopy and Atomic Force Microscopy (AFM) to investigate the vibrational signatures and morphometric parameters of MRC-5 pulmonary fibroblasts and two melanoma cell lines: SK-MEL-19 (less aggressive) and SK-MEL-103 (more aggressive). Raman spectra from melanoma cells exhibited increased intensities in bands associated with structural and signaling amino acids, indicative of extracellular matrix remodeling, cyto- and cytoskeletal reorganization, and apoptotic deregulation. SK-MEL-104 showed more pronounced proline and guanine peaks, consistent with its higher aggressiveness and known BRAF and NRAS mutations. AFM topography revealed marked crater-like features in nuclear and cytoplasmic regions, suggestive of cytoskeletal alterations. Measurements of relative surface area and nucleus-to-cytoplasm ratio confirmed the lower metabolic activity of MRC-5 fibroblasts compared with the high plasticity and metabolic activity of melanoma cells. These results provide structural and biochemical insights into melanoma adaptation and proliferation within lung tissue and underscore the complementary potential of Raman spectroscopy and AFM as powerful tools for characterization and prognosis in metastatic progression studies.</p>