In the present work, the antimicrobial, antioxidative, antigenotoxic and anticancer potentials of methanol and ethanol extracts of Ectomycorrhizal mushroom (ECM) Pisolithus arhizus was evaluated. In vitro antibacterial activity was tested by minimum inhibitory concentration (MIC). MIC values of the extracts showed that the antimicrobial effect of the methanol extracts was higher than the ethanol extracts effect on clinical isolates. The antioxidant potential was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, metal chelating, total phenolic content, total flavonoid content, beta-carotene and lycopene components and total antioxidant activity assays. The methanol extracts of mushroom had the highest total phenolic, total flavonoid contents and total antioxidant capacity. Flavonoid analysis of the mushroom extracts has been carried out by high-performance liquid chromatography (HPLC). According to the results, we detected that the methanol extracts had higher luteolin, rutin, quercetin and catechin amount. The antigenotoxi-city of the mushroom was established by Comet test. As a result of this study, both two extracts of P. arhizus were also observed to decrease the effect of H2O2 on human lymphocytes that cause DNA damage. In addition, the cytotoxicity experiments were conducted with the WST-1 Cell proliferation assay kit and HT29 cell. The ethanol and methanol extracts of mushroom sample have been found to have a high cytotoxicity on colon cancer cells and this effect increased proportionally with their increasing concentration. The data of present study demonstrated that ECM P. arhizus may be utilized as prosing sources of therapeutics because of its effective antimicrobial, antioxidant anticancer properties.