This research work introduces a study about mushroom cultivation, biochemical analysis, and antioxidant performance. The cultivation of edible oyster mushroom Pleurotus floridanus (EMPF) was carried out utilizing a hanging technique based on different substrate cultures including rice straw, wheat straw, a mixture of rice and wheat straw, a mixture of agricultural lime and wheat straw, a mixture of agricultural lime and rice straw, a mixture of quicklime and rice straw. After that, this research work analyzed the carbohydrate and protein contents in addition to investigating the antioxidant performance of EMPF as a species of oyster mushroom to improve awareness of the beneficial impact of EMPF in commercial markets. Based on the obtained results of biochemical analysis, the investigated EMPF (fresh) has 4.979 g/ 100 g carbohydrate using the traditional anthrone test. Additionally, the studied fresh EMPF has 2.413 g/ 100 g as a total protein content using the Folin-Ciocalteu test. Besides, FT-IR indicates the chemical existence of function groups of protein and carbohydrate in the investigated EMPF. The main contents of the cultivated EMPF are moisture followed by carbohydrates and protein in addition to traces from ash and metals. The antioxidant of the cultivated EMPF was evaluated via 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity values using different mushroom concentrations from 30 -150 g/L indicating that the optimum one is 90 g/L. The antimicrobial of the cultivated EMPF was tested and the results affirm the high performance of the EMPF as antibacterial or antifungal compared with familiar antibacterial or antifungal (Streptomycin or fluconazole, respectively). To conclude, this work introduces the antimicrobial and antioxidant of the studied EMPF.