This study was designed to understand the effect of extraction temperature, i.e., room temperature (GLRT), 50°C (GL50), 100°C (hot water; GL100), and 200°C (GL200) on antioxidant and biological activity of G. lucidum. The % yield obtained was 5.3%, 7.6%, 10.7%, and 13.2% at various extraction temperatures; room temperature, 50°C, 100°C and 200°C, respectively. Similarly, phenolic content (51.6, 57.9, 82.9, and 93.1 mg/g extract) and flavonoid content (18.8, 23.2, 34.3, and 36.3 mg/g extract) were observed to be increased with rise in extraction temperature. However, extraction temperature resulted in loss of antioxidant activities above 100°C as evident by chemical assays such as DPPH, FRAP, ABTS, and TRP conducted on extracts. In contrast, three bioactive compounds, i.e., adenine (3.26, 3.48, 2.16, and 1.45 mg/g extract), uracil (3.99, 3.21, 2.51, and 1.47 mg/g extract), and adenosine (5.92, 5.62, 2.22 and 0.7 mg/g extract), quantified by high performance thin layer chromatography showed decrease in their content with increasing extraction temperature. Extract prepared at room temperature and 50°C prevented loss of cell viability and generation of reactive oxygen species resulted after hydrogen peroxide exposure; however, cytoprotective efficacy was not significant at 100°C and 200°C The order of cytoprotective effects observed by these extract were in the following order: room temperature ≥ 50°C > 100°C > 200°C. Overall, the optimal temperature conditions for the efficient extraction of G. lucidum with water retaining bioactive compounds and biological activity was found to be below 100°C.