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Research Background: In the recent years, considerable attention has been given to selenium status since its deficiency is linked with various disorders and affects at least 13% of world population. Additionally, mushrooms are known to possess pronounced capacity for absorption of various micronutrients, including Se, from soil/substrate. Here, we investigate the possibility of using Se-rich zeolitic tuff as a supplement for production of selenized mushroom. Furthermore, the impact of the enrichment on the activity of antioxidant enzymes and biological potential of Coriolus versicolor medicinal mushroom is studied., Experimental Approach: Se(IV)- and Se(VI)-modified natural zeolitic tuff from the Serbian deposit Zlatokop was used as supplement for mushroom cultivation. To examine the effectiveness of selenium enrichment, we determined total selenium with inductively coupled plasma mass spectrometry (ICP-MS), together with the activity of antioxidant enzymes in fresh fruiting bodies and biological potential of methanolic extracts. Antioxidant activity was evaluated using the appropriate tests for: inhibition of lipid peroxidation, DPPH free radical scavenging assay, Fe(III)-reducing antioxidant power assay and ability of chelating Fe 2+ ions. The antibacterial activity against foodborne pathogens was measured by broth microdilution assay. Additionally, chemical composition of the prepared extracts was studied using UV-Vis and Fourier transform infrared (FTIR) spectroscopy., Results and Conclusions: Content of selenium detected in biofortified C. versicolor was even 470 times higher than in control on dry mass basis ((140.7±3.8) vs (0.3±0.1) µg/g), proving that Se-rich zeolitic tuff is an excellent supplement for mushroom production. Furthermore, the results of monitoring the activity of antioxidant enzymes revealed that most of the Se-enriched mushrooms exhibited higher superoxide dismutase (SOD) and catalase (CAT) and lower glutathione peroxidase (GSH-Px) activities than control. Due to higher amounts of enzymes, which can quickly catalyze the reduction of superoxide radicals, the quality of selenium-enriched mushrooms is preserved for a longer period of time. Investigation of biological potential indicated that Se-enriched mushroom methanolic extracts, generally, expressed enhanced antioxidant properties. Additionally, extracts showed antibacterial activity against all tested pathogenic microorganisms., Novelty and Scientific Contribution: Cultivation of mushrooms on Se-enriched zeolitic tuff is a new technological approach for obtaining Se-fortified food/supplements with enhanced antioxidant and antibacterial activities., Competing Interests: CONFLICT OF INTEREST The authors declare that they have no conflict of interest.