The effects of different cooking methods (steaming, boiling, air frying, and oven baking) and cooking times (0, 5, 10, 15, and 20 min) on the bioactive components (total phenol, total flavonoid, crude polysaccharides, and eritadenine), color, texture, microstructure, and volatiles in shiitake mushrooms were investigated in this study. Steaming, boiling, and air frying for 5–20 min could decrease the contents of all the four bioactive components in the shiitake mushroom. However, oven baking for 5 min and 10 min showed the highest contents of total phenolics and total flavonoids, respectively. Moreover, the lowest losses of crude polysaccharides and eritadenine were observed for oven baking for 5 min and 15 min, respectively. The lightness of shiitake mushrooms was decreased by all treatments; however, steaming could keep a higher brightness compared with other methods. The microstructure was damaged by all cooking methods, especially air frying for 20 min. Meanwhile, steaming for 20 min decreased the hardness mostly, and there was no significant difference with air frying for 20 min. All cooking treatments decreased the complexity of the flavors and the relative contents of volatile compounds; the lowest contents were found when boiling for 5 min. From these results it can be seen that the physical, histological, and chemical features in shiitake mushroom were influenced by cooking methods and times. In addition, our results provide valuable information for the cooking and processing of shiitake mushrooms and other fungi.