It was recently demonstrated that newly invented positronium imaging may be used for improving cancer diagnostics by providing additional information about tissue pathology with respect to the standardized uptake value currently available in positron emission tomography (PET). Positronium imaging utilizes the properties of positronium atoms, which are built from the electrons and positrons produced in the body during PET examinations. We hypothesized that positronium imaging would be sensitive to the in vitro discrimination of tumor-like three-dimensional structures (spheroids) built of melanoma cell lines with different cancer activities and biological properties. The lifetime of ortho-positronium (o-Ps) was evaluated in melanoma spheroids from two cell lines (WM266-4 and WM115) differing in the stage of malignancy. Additionally, we considered parameters such as the cell number, spheroid size and melanoma malignancy to evaluate their relationship with theo-Ps lifetime. We demonstrate pilot results foro-Ps lifetime measurement in extracellular matrix-free spheroids. With the statistical significance of two standard deviations, we demonstrated that the higher the degree of malignancy and the rate of proliferation of neoplastic cells, the shorter the lifetime of ortho-positronium. In particular, we observed the following indications encouraging further research: (i) WM266-4 spheroids characterized by a higher proliferation rate and malignancy showed a shortero-Ps lifetime than WM115 spheroids characterized by a lower growth rate. (ii) Both cell lines showed a decrease in the lifetime ofo-Ps after spheroid generation on day 8 compared to day 4 in culture, and the meano-Ps lifetime was longer for spheroids formed from WM115 cells than for those formed from WM266-4 cells, regardless of spheroid age. The results of this study revealed that positronium is a promising biomarker that may be applied in PET diagnostics for the assessment of the degree of cancer malignancy.