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The effect of heat treatment on dried fruiting bodies of Reishi medicinal mushroom (<i>Ganoderma lingzhi</i>) is investigated. Control and samples treated for 20 min at temperatures of 70, 120, 150 and 180 °C were subjected for their free radical scavenging capacity, different glucans and total phenolic content determination. The growth rate of <i>Escherichia coli</i> and <i>Lactobacillus casei</i> supplemented with control and heat-treated samples is also investigated. The roasted mushroom samples at 150 °C and 180 °C showed the highest level of β-glucan (37.82%) and free radical scavenging capacity on 2,2-diphenyl-1-picrylhidrazyl (DPPH•) and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS•+). The content of total phenolics (TPC) was also influenced by heat treatment and significantly higher TPC values were recorded in samples treated at 120 °C and 150 °C. The presence of reducing sugars was only detected after heat treatment at 150 °C (0.23%) and at 180 °C (0.57%). The heat treatments at 120, 150 and 180 °C, significantly attenuated the number of colony-forming units (CFU) of pathogenic <i>E. coli</i>, in a linear relationship with an elevated temperature. The supplementation of heat-treated Reishi mushroom at 120 °C resulted in the highest growth rate of probiotic <i>L. casei</i>. The obtained results in this study revealed the significant effect of short-term heat treatment by enhancing the antioxidant capacity, β-glucan solubility and prebiotic property of the dried basidiocarp of Reishi mushroom.