Pleurotus ostreatus (Oyster mushroom) ranks as the second most cultivated edible mushroom globally after Agaricus bisporus, known for its ecological and medicinal benefits. This study aimed to investigate the impact of various substrates on the growth and biological efficiency of Oyster mushrooms. Materials and Methods: Different substrates including Wheat straw, Groundnut straw, Soybean straw, and various sizes of sawdust were sterilized in boiling water at 120°C for 1 hour prior to spawn inoculation. The experiment was conducted in a controlled environment with humidity maintained at 80-85% in a dark room. Results: Throughout the growth period, various morphological characteristics such as spawn germination time, mycelium formation, pinhead formation, fruiting body formation time, and biological efficiency of Oyster mushrooms were evaluated. The study highlighted that Groundnut substrate exhibited rapid growth in terms of spawn germination, mycelium growth, pinhead formation, and fruiting body development. Additionally, Soybean substrate demonstrated the highest biological efficiency and moisture content.