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The innovative strategy of using nanoparticles in radiotherapy has become an exciting topic due to the possibility of simultaneously improving local efficiency of radiation in tumors and real-time monitoring of the delivered doses. Yttrium oxide (Y 2 O 3 ) nanoparticles (NPs) are used in material science to prepare phosphors for various applications including X-ray induced photodynamic therapy and in situ nano-dosimetry, but few available reports only addressed the effect induced in cells by combined exposure to different doses of superficial X-ray radiation and nanoparticles. Herein, we analyzed changes induced in melanoma cells by exposure to different doses of X-ray radiation and various concentrations of Y 2 O 3 NPs. By evaluation of cell mitochondrial activity and production of intracellular reactive oxygen species (ROS), we estimated that 2, 4, and 6 Gy X-ray radiation doses are visibly altering the cells by inducing ROS production with increasing the dose while at 6 Gy the mitochondrial activity is also affected. Separately, high-concentrated solutions of 25, 50, and 100 µg/mL Y 2 O 3 NPs were also found to affect the cells by inducing ROS production with the increase of concentration. Additionally, the colony-forming units assay evidenced a rather synergic effect of NPs and radiation. By adding the NPs to cells before irradiation, a decrease of the number of proliferating cell colonies was observed with increase of X-ray dose. DNA damage was evidenced by quantifying the γ-H2AX foci for cells treated with Y 2 O 3 NPs and exposed to superficial X-ray radiation. Proteomic profile confirmed that a combined effect of 50 µg/mL Y 2 O 3 NPs and 6 Gy X-ray dose induced mitochondria alterations and DNA changes in melanoma cells.