Background: Cancer cells may develop resistance to radiation which results in decreasing effects of radiation therapy. The effectiveness of high dose radiation can be increased with previously delivered low dose. Also, the approach that has been used to enhance the efficacy of radiation is to increase the radiosensitization. The focus of interest is on usage of gold-based nanoparticles as radiosensitizers. Modification of gold nanoparticle surface may increase its effectiveness in cells. The aim of the study was to prepare and characterize nanogold/β-cyclodextrin formulation (nAu/β-CD) and to investigate cytotoxicity and differences in radio-adaptive response of irradiated normal and malignant cell lines. Materials and Methods: Nanoparticles distribution and zeta-potential of nAu/β-CD were obtained by dynamic light scattering (DLS). Cell lines MRC-5 and A549 were pretreated with 0.05 µM nAu/β-CD formulation and irradiated in single and double regimes. The MTT test was performed after 24 h and 72 h of recovery.  Results: Nanogold particles didn’t express cytotoxic effect on MRC-5 and A549 cells. Tested nanoformulations with nAu/β-CD ratio 1:1 achieved the best cytotoxic effect against A549 cell line. Pre-treatment with low dose (0.05 Gy) prior to therapeutic dose (2 Gy) and application of nAu/β-CD nanoformulation decreased the cell survival in all investigated samples, showing diverse effects on normal and tumor cells. Conclusion: Results indicate potential selectivity and increased efficiency of both applied radiation and pre-treatment with nAu/β-CD regarding the malignant cells, while sparing the normal tissue from radiation damage, which could be beneficial for the radiotherapy.